Cycloalkyl derivatives of 3-hydroxy-4-pyridinones

ABSTRACT

The present invention provides an cycloalkyl derivative of 3-hydroxy-4-pyridinone which is useful for the chelation of metal ions such as iron. Its preparation and use is described. In particular, the invention concerns the removal of iron in chemical and biological systems including chelating agents having the formula (I); wherein R 1  is X with the proviso that R 2  is Y; or R 1  is T with the proviso that R 2  is W; or R 1  is X with the proviso that R 2  R 5  N when taken together form a heterocyclic ring selected from piperidinyl, morpholinyl, pyrrolidinyl or piperazinyl, wherein the group piperidinyl, morpholinyl, pyrrolidinyl or piperazinyl is either unsubstituted or substituted with one to three C 1  to C 6  alkyl groups. X is C 3 -C 6  cycloalkyl; Y is selected from the group consisting of C 1  to C 6  cycloalkyl; C 1  to C 6  alkyl, and C 1  to C 6  alkyl monosubstituted with a C 3 -C 6  cycloalkyl; T is C 1  to C 6  alkyl; W is C 3 -C 6  cycloalkyl; R 3  is selected from the group consisting of hydrogen and C 1  to C 6  alkyl; R 4  is selected from the group consisting of hydrogen and C 1  to C 6  alkyl; R 5  is selected from the group consisting of hydrogen and C 1  to C 6  alkyl; and its pharmaceutically acceptable salt thereof. Pharmaceutical compositions of such compounds are useful in the removal of excess body iron from patients with iron overload diseases.

FIELD OF THE INVENTION

The invention relates to novel 3-hydroxy-4-pyridinone derivatives andtheir use in chelating ferric (III) ions. More particularly, theinvention relates to cycloalkyl derivatives of 3-hydroxy-4-pyridinone.

BACKGROUND

3-Hydroxy-4-pyridinones are bidentate ligands that chelate to theFe(III) ion in the ratio of 3:1 and are useful in the removal of excessbody iron in humans. Iron overload may be due to excess dietaryconsumption of iron, inherited genetic conditions such ashaemochromatosis and regular blood transfusion. Such transfusions areused to treat medical conditions such as thalassaemia, sickle cellanaemia, idiopathic haemochromatosis and aplastic anaemia. Increasediron absorption from transfusion leads to iron overload. Upon saturationof ferritin and transferrin in the body, iron deposit in many tissuessuch as the myocardium, liver and endocrine organs resulting in toxiceffects.

The scope of iron chelator research and the proposed utility ofchelators have been reviewed (Current Medicinal Chemistry, 2003, 10,983-985, Tim F. Tam, et al). Iron chelators may be useful to preventhydroxy radical formation, treatment of cancer, malaria, post-ischaemicreperfusion, and neurodegenerative diseases. Iron chelators such asDesferal™ (desferrioxamine mesylate) and Ferriprox™ (deferiprone) areused to remove excess body iron in thalassemia major patients becausethe human body has no effective means to excrete the iron accumulatedfrom blood transfusion. Desferrioxamine is administered daily bysubcutaneous infusion over a period of 8 to 12 hours. At present,deferiprone (1,2-dimethyl-3-hydroxy-4-pyridinone) is the only orallydrug available. It undergoes extensive metabolism in the liver and morethan 85% of the administered dose is recovered in the urine as thenon-chelating O-glucuronide (Drug Metab. Dispo. 1992, 20(2), 256-261, S.Singh, et al.). A relatively high oral dose of 75 mg/kg (3.5 to 4 gm perday) is required for the treatment of iron overload conditions.Therefore, there is a need to identify a new orally activehydroxypyridinone with improved pharmacological properties thandeferiprone.

Voest et. al. (Annals of Internal Medicine 1994, 120, 490-499) reviewedthe clinical experience of iron chelators in non-iron overloadedconditions. Iron chelators were used to produce antioxidant effects,antiproliferative effects, antiprotozal effects and for aluminumchelation, and may be used be for a variety of disease state such as thetreatment of rheumatoid arthritis, the protection against anthracyclinecardiac poisoning, for limiting mycocardial ischemia-reperfusion injury,as antitumour agents, and for the treatment of malaria. In addition, vanAsbeck B. S. et. al. (J Clin Virol. 2001 February;20(3):141-7) reportedthat iron chelators have anti-HIV activities. Therefore the utitlies ofiron chelators are not only restricted to the treatment ofiron-overloaded conditions.

The members of the 3-hydroxy-4-pyridinones class are known for theirability to chelate iron. Prior art includes RE 35,948, U.S. Pat. Nos.6,448,273, 6,335,353 and 5,480,894. In U.S. Pat. No. 6,335,353, theester prodrug derivatives of 3-hydroxy-4-pyridinones are used tofacilitate efficient iron extraction from the liver, however none of thedesigned compounds has reached evaluation in humans.

In other approaches, selected new compounds were designed to block thephase II metabolism of O-glucuronidation at the C3 oxygen of thedeferiprone skeleton. U.S. Pat. No. 5,688,815 reported1-alkyl-3-hydroxy-4-pyrdinones with a C2 methyl group substituted with aphenyl or heteroyl ring and a hydroxy group, and the N1 substituentbeing a lower alkyl. U.S. Pat. No. 6,335,353 described1-alkyl-3-hydroxy-4-pyridinone with a C2 alkylcarbamoyl, arylcarbamoyl,or an aralkylcarbamoyl group and the N1-substituent is an aliphatichydrocarbon group. The use of C2-methylcarbamoyl functionality incompound such as CP502(1,6-Dimethyl-3-hydroxy-4(1H)-pyridinone-2-carboxy-(N-methyl)-amidehydrochloride; U.S. Pat. No. 6,335,353) effectively blocked theO-glucuronidation at the C3 oxygen. Other analogues in U.S. Pat. No.6,335,353 include CP506(1,6-Dimethyl-3-hydroxy-4(1H)-pyridinone-2-carboxy-(N-isopropyl)-amidehydrochloride), the C2-isopropylcarbamoyl analogue and CP508(1,6-Dimethyl-3-hydroxy-4(1H)-pyridinone-2-carboxy-(N,N-dimethyl)-amidehydrochloride), the dimethylcarbamoyl analogue. CP502, CP506 and CP508are prior art and have not been evaluated in humans.

SUMMARY OF THE INVENTION

A first aspect of the present invention provides a3-hydroxypyridin-4-one compound of formula I inclusive of apharmaceutically acceptable salt of the compound of formula I,

-   -   wherein:    -   R¹ is X with the proviso that R² is Y; or    -   R¹ is T with the proviso that R² is W; or    -   R¹ is X with the proviso that R²R⁵N when taken together, form a        heterocyclic ring selected from piperidinyl, morpholinyl,        pyrrolidinyl or piperazinyl, wherein the group piperidinyl,        morpholinyl, pyrrolidinyl or piperazinyl is either unsubstituted        or substituted with one to three C₁-C₆ alkyl groups;        -   X is C₃-C₆ cycloalkyl;        -   Y is selected from the group consisting of C₃-C₆ cycloalkyl,            C₁ to C₆ alkyl and C₁ to C₆ alkyl monosubstituted with a            C₃-C₆ cycloalkyl;        -   T is C₁ to C₆ alkyl;        -   W is C₃-C₆ cycloalkyl;    -   R³ is selected from the group consisting of hydrogen and C₁-C₆        alkyl;    -   R⁴ is selected from the group consisting of hydrogen and C₁-C₆        alkyl; and    -   R⁵ is selected from the group consisting of hydrogen and C₁-C₆        alkyl.

A second aspect of the present invention provides use of a compound offormula I in the treatment of iron overload related disease.

A third aspect of the invention provides a pharmaceutical compositioncomprising a compound of formula I.

One preferred class of compounds of this invention is the compound offormula I wherein R¹ is X with the proviso that R² is Y. X is C₃-C₆cycloalkyl; Y is C₁ to C₆ alkyl; R³ is hydrogen; R⁴ is C₁-C₆ alkyl andR⁵ is hydrogen.

A still more preferred compound under this subset is a compound offormula I wherein R⁴ is methyl, X is cyclopropyl and Y is methyl and thecompound is1-cyclopropyl-3-hydroxy-6-methyl-4-oxo-1,4-dihydro-pyridine-2-carboxylicacid methylamide.

A second preferred class of compounds of this invention is a compound offormula I wherein R¹ is X with the proviso that R² is Y, X is C₃-C₆cycloalkyl, Y is C₃-C₆ cycloalkyl, R³ is hydrogen, R⁴ is C₁-C₆ alkyl andR⁵ is hydrogen.

A preferred compound within this subset is a compound wherein R⁴ ismethyl, X═Y=cyclopropyl and the compound is1-cyclopropyl-3-hydroxy-6-methyl-4-oxo-1,4-dihydro-pyridine-2-carboxylicacid cyclopropylamide.

A third preferred class of compounds of formula I is a compound whereinR¹ is T with the proviso that R² is W; T is C₁ to C₆ alkyl; W is C₃-C₆cycloalkyl, R³ is hydrogen, R⁴ is C₁-C₆ alkyl and R⁵ is hydrogen.

A more preferred compound under this subset is a compound wherein R⁴ ismethyl, T is methyl and W is cyclopropyl, the compound is3-hydroxy-1,6-dimethyl-4-oxo-1,4-dihydro-pyridine-2-carboxylic acidcyclopropylamide.

A fourth preferred class of compounds of this invention is a compound offormula I wherein R¹ is X with the proviso that R² is Y, X is C₃-C₆cycloalkyl; Y is C₁ to C₆ alkyl; R³ is hydrogen; R⁴ is C₁-C₆ alkyl, R⁵is methyl.

A still more preferred compound under this subset is a compound offormula I wherein R⁴ is methyl, X is cyclopropyl and Y is methyl, andthe compound is1-cyclopropyl-3-hydroxy-N,N,6-trimethyl-4-oxo-1,4-dihydropyridine-2-carboxamide.

The most preferred compounds of this invention are compound IA, acompound of formula I wherein R³═H, R⁴=methyl, with the proviso thatR¹═X=cyclopropyl, R²═Y and Y is selected from the group C₃-C₆cycloalkyl; C₁ to C₆ alkyl; C₁ to C₆ alkyl monosubstituted with a C₃-C₆cycloalkyl; or R¹═X=cyclopropyl, R²R⁵N when taken together form aheterocyclic ring selected from piperidinyl, morpholinyl, pyrrolidinylor piperazinyl, wherein the group piperidinyl, morpholinyl, pyrrolidinylor piperazinyl is either unsubstituted or substituted with one to threeC₁-C₆ alkyl groups.

In light of the above, the present invention provides a cycloalkylderivative of 3-hydroxy-4-pyridinone having improved properties ascompared to compounds reported in the prior art. The cycloalkyl group isattached to the N1 and/or C2 amido N atom. Prior to this application,compounds with N1-cycloalkyl substituent or C2 amido N-cycloalkylsubstituent were unknown in the literature. These compounds are notprodrugs and have excellent metal ion selectivity. They show nocomplexation with essential metals such as calcium and magnesium at pH7.4 in chemical assays. The D_(7.4) value is within the range of anestablished drug deferiprone and the compound is orally active in theiron overload rat model. These compounds are designed with favorablephenolic C3 OH pKas in the range of 8.3 to 8.8, a pFe³⁺ value of above20, a smooth 1:3 ferric chelate formation as evident by Job's plot, anda D_(7.4) value>0.1. The single crystal structure of the Fe(III) chelateconfirms that the compound of formula I is a bidentate ligand.

BRIEF REFERENCE TO THE DRAWINGS

FIG. 1 is a diagrammatic representation of Job's plot of Apo6622(1-cyclopropyl-3-hydroxy-6-methyl-4-oxo-1,4-dihydro-pyridine-2-carboxylicacid cyclopropylamide), a compound of formula I.

FIG. 2 is a diagrammatic representation of Job's plot of Apo6617(1,6-dimethyl-3-hydroxy-4-oxo-1,4-dihydro-pyridine-2-carboxylic acidcyclopropylamide), a compound of formula I.

FIG. 3 is a diagrammatic representation of Job's plot of Apo6619(1-cyclopropyl-3-hydroxy-6-methyl-4-oxo-1,4-dihydro-pyridine-2-carboxylicacid methylamide), a compound of formula I.

FIG. 4 is a speciation plot for Fe³⁺-Apo6619.

FIG. 5 is a speciation plot for Fe³⁺-Apo6617.

FIG. 6: Effectiveness of Apo6619 and Apo6617 in Promoting Urinary IronExcretion in the Iron Overloaded Rat (n=6).

FIG. 7: Single crystal structure of Fe(Apo6617)₃ chelate.

FIG. 8: Single crystal structure of Fe(Apo6619)₃ chelate.

FIG. 9: Cyclic voltammogram of Fe-Apo6619 system at pH 7.4.

TABLE 1: Chemical properties of compound of formula I.

TABLE 2: Metal binding selectivity of Apo6619.

TABLE 3: Effectiveness of Apo6619 and Apo6617 in Promoting Fecal IronExcretion in the Iron Overloaded Rat (n=6). Values are expressed asμg/day/kg.

TABLE 4: Effectiveness of Apo6619 and Apo6617 in Promoting Urinary andFecal Iron Excretion in the Iron Overloaded Rats (n=6/group). Values areexpressed as μg/day/kg. Fecal excretion values 3 days after chelatoradministration are given and compared to the baseline values determined3 days prior to chelator administration. Values are expressed asmean±1SD.

TABLE 5: Crystal data and structure refinement for Fe(Apo6617)₃.

TABLE 6. Bond lengths [Å] and angles [°] for Fe(Apo6617)₃.

TABLE 7: Crystal data and structure refinement for Fe(Apo6619)₃.

TABLE 8. Bond lengths [Å] and angles [°] for Fe(Apo6619)₃.

DETAILED DESCRIPTION OF THE INVENTION

As used herein:

Alkyl means a branched or unbranched saturated hydrocarbon chain having,unless otherwise noted, one to six carbon atoms, including but notlimited to methyl, ethyl, propyl, isopropyl, n-propyl, butyl, sec-butyl,isobutyl, n-pentyl, hexyl.

The term “cycloalkyl” as employed herein alone or as part of anothergroup includes saturated cyclic hydrocarbon groups containing 1 ring,including monocyclic alkyl, containing a total of 3 to 6 carbons formingthe ring, which includes cyclopropyl, cyclobutyl, cyclopentyl andcyclohexyl.

Pharmaceutically acceptable, non-toxic salts refer to pharmaceuticallyacceptable salts of the compounds of this invention, which retains thebiological activity of the parent compounds and are not biologically orotherwise undesirable (e.g. the salts are stable). Salts of the twotypes may be formed from the compounds of this invention: (1) Salts ofinorganic and organic bases from compounds of formula I, which has aphenol functional group, and (2) Acid addition salts may be formed atthe amine functional group of compounds of formula I of this invention.

Pharmaceutically acceptable salts derived from inorganic bases includesodium, potassium, lithium, ammonium, calcium, and magnesium salts.Particularly preferred are the sodium, calcium and magnesium salts.Pharmaceutically acceptable, non-toxic salts derived from organic basesinclude salts of primary, secondary and tertiary amines, substitutedamines including naturally occurring substituted amines, cyclic aminesand basic ion exchange resins. Such salts are exemplified by, forexample, 2-amino-2-hydroxymethyl propane 1,3-diol, isopropopylamine,tromethamine, glucosamine, methylglucamine, purines, piperazine,piperidine, N-ethylpiperidine, polyamine resins and the like.

Pharmaceutically acceptable acid addition salts are formed withinorganic and organic acids such as halo acids, sulfuric acid, nitricacid, phosphoric acid, methanesulfonic acid, and ethanesulfonic acid.

The compounds of this invention are 2-amido derivatives of4-oxo-1,4-dihydropyridine-2-carboxamide derivatives having the generalstructure:

Most compounds are named as a derivative of4-oxo-1,4-dihydropyridine-2-carboxamide, for example:

1-cyclopropyl-N-hexyl-3-hydroxy-6-methyl-4-oxo-1,4-dihydropyridine-2-carboxamide

N-cyclohexyl-1-cyclopropyl-3-hydroxy-6-methyl-4-oxo-1,4-dihydropyridine-2-carboxamide

N-(cyclohexylmethyl)-1-cyclopropyl-3-hydroxy-6-methyl-4-oxo-1,4-dihydropyridine-2-carboxamide

In some cases, the compounds are named using “pyridin-4(1H)-one” as thebasic skeleton. Examples are:

1-cyclopropyl-3-hydroxy-6-methyl-2-(morpholin-4-ylcarbonyl)pyridin-4(1H)-one

1-cyclopropyl-3-hydroxy-6-methyl-2-[(4-methylpiperazin-1-yl)carbonyl]pyridin-4(1H)-one

The term “animals” refers to humans as well as all other animal species,particularly mammals (e.g. dogs, cats, horses, cattle, pigs, etc.),reptiles, fish, insects and helminths.

Compounds of this invention are designed to improve properties to knowndeferiprone analogues. One criteria used in the design rationale of oralactive chelators in the 3-hydroxy-4-pyridinone series are compoundshaving pFe³⁺ values higher than deferiprone (pFe³⁺=19.7). The definitionof pFe³⁺ used herein is the concentration of ferric ion in solution whenthe total amount of iron equals 10⁻⁶ M and the concentration of ligandis 10⁻⁵ M and pH is 7.4. It is calculated using experimental determinedpKa and metal complexation constants using Hyperquad software (Version2.1, Peter Gans, University of Leeds). The lowering of the pKa value ofthe C3 phenolic OH to less than 8.8 ensures that a higher pFe³⁺ valuewhen combined with a favorable complexation constant β₃. The concept ofcomplexation is detailed below.

The stepwise and overall complexation constants of a bidentate ligandsuch as 3-hydroxy-4-pyridinone follow:Fe(III)+Ligand→Fe[Ligand]₁  K₁Fe[Ligand]₁+Ligand→Fe[Ligand]₂  K₂Fe[Ligand]₂+Ligand→Fe[Ligand]₃  K₃

-   -   Complexation constant β₃=K₁.K₂.K₃

The iron chelator drug deferiprone (1,2-dimethyl-3-hydroxy-4-pyridinone)chelates iron with a complexation constants (log β₃) of 36.3 and a pFe³⁺of 19.7. The pKas of deferiprone is 3.56 and 9.64. Most compounds ofthis invention have similar complexation constants (log β₃) in the rangeof 34 to 36, a pFe³⁺>20 and favorable phenol pKa values of 8.3 to 8.8.Accordingly, compounds of this invention are excellent chelators ofFe(III). The theoretically calculated human jejunum effectivepermeability [P_(eff)] of compounds of this invention is predicted bycomputational calculations using QMPRPlus™ software (from Simulationplusinc.). Most compounds of this invention have calculated P_(eff) in therange of 1±0.3 (cm/s×10⁻⁴), implying that the compounds have good humanjejunal permeability. The chemical properties of representativecompounds of formula I are shown in Table 1.

Significantly, compounds of formula I with cycloalkyl groups at R¹and/or R² are metal chelators with high pFe³⁺ values. The D_(7.4) valuesof compounds of formula I are similar to deferiprone and further studiesin iron overload rats showed that compounds of formula I are effectivein the removal of iron in vivo. The details of the animal efficacy studyare shown in the examples below.

Compounds of formula I do not bind essential metals such as manganese,calcium and magnesium. The pM values and complexation constants of arepresentative compound of formula I are shown in Table 2 (and discussedin more detail in example 11). The compound has preference for bindingFe³⁺ over other bivalent and trivalent metals such as Cu, Zn and Al.

Compounds of formula I are novel cycloalkyl derivatives of3-hydroxy-4-pyridinones. They have pFe³⁺ values above 20, a favorableD_(7.4) value comparable to deferiprone, a preference towards thechelation of Fe³⁺ and a C2-alkylcarbamoyl or C2-cycloalkylcarbamoylmoiety that is designed to block the phase II metabolism of the 3-OHgroup.

In addition to the above, compounds of formula I binds Fe³⁺ in the ratioof 1:3 at physiological conditions at pH 7.4. The Job's plot analysisconfirms the 1:3 ratio of chelator to ferric metal (FIG. (1 to 3) andexample 9).

The speciation plots of the Fe-complex vs. different pHs can becalculated by using Hyperquad Stimulation and Speciation software(HYSS2© 2000 Protonic Sofware) with the input of experimental pKas(example 10 and 11) and the complexation constants K₁, K₂ and K₃(example 14). FIGS. 4 and 5 illustrate the speciation plot of compoundsof formula I at different pHs. In both studies, representative compoundsof formula I exclusively form FeL₃ chelates at pH above 7.0 (where L isa bidentate ligand), thus ensuring no presence of FeL₂ ⁺ or FeL²⁺species at physiological pH. The absence of these species ensures thatthere is no exposed iron in-vivo at the physiological pH of 7.4.

Compounds of formula I wherein R¹ is X with the proviso that R² is Y; orR¹ is T with the proviso that R² is W is prepared according to themethod outlined in Scheme 1.

Acid (II) is reacted with 1,1′-carbonyldiimdazole in an inert solventfor 2 to 5 hrs, preferably 5 hrs in an inert solvent at temperaturesbetween 30 to 70° C. Followed by the addition of an amine R²R⁵NH, thecompound (III) is isolated by conventional means. A solution of (III)and an amine R¹NH₂ in an inert solvent such as an alcohol is heated at50 to 80° C. to effect the amine insertion of (III) for a period of 3 to48 hrs to give compound (IV). An alternate method for the preparation ofcompound (IV) involves the reaction of a compound of formula (II)) withan amine R¹NH₂ in an inert solvent to give the acid of formula (V).Compound (V) is then reacted with thionyl chloride and dimethylformamideto give a compound of formula (IV). The compound is isolated bytraditional means eg. column chromatography and crystallization.Hydrogenation of compound (IV) in alcohol over a hydrogenation catalystaffords compound (I), which is isolated by conventional means. Thepreferred hydrogenation catalyst is palladium on carbon or palladiumhydroxide on carbon and Raney Ni. The preparation of the startingmaterial acid (II) is reported in U.S. Pat. No. 6,426,418. A generalprocedure for the preparation of an acid of formula (V) can be found inCA 2379370.

Compounds of formula I were tested in iron overloaded rats. The fecaliron excretion and urinary excretion data for representative compoundsApo6617 and Apo6619 are shown in Tables 3 and 4, and FIG. 6,respectively. Both compounds showed significant fecal iron excretionwhen compared to control at an oral dose of 113 and 450 μmol/kg.Further, Apo6619 and Apo6617 facilitate the urinary excretion of ironsignificantly higher than deferiprone at 450 μmol/kg. Both compounds areconsidered more potent than deferiprone in iron mobilization in ironoverloaded rats.

The ferric chelate of compounds of formula I have been synthesized andisolated (example 16). The single crystal structures of Fe(Apo6617)₃ andFe(Apo6619)₃ definitively prove that these bidentate compounds reactedwith Fe(III) to give a 1:3 trisbidentate chelate (Table 5 to 8, FIG.7-8).

Another criteria in the design of compounds of formula I concernscontrolling the redox potential of the Fe-chelate system at pH 7.4 to anegative value below −320 mv (vs NHE) to prevent any reactions withoxygen species. Iron exists in multiple states including Fe²⁺ and Fe³⁺.The iron (II)/iron (III) pair can act as a pair of one electron reducingagent and oxidizing agent. According to Crumbliss(http://www.medicine.uiowa.edu/FRRB/VirtualSchool/Crumbliss-Fe.pdf) andPierre (BioMetals, 12, 195-199, 1999), selective chelation of iron withredox potential control is a means to prevent iron from participating ina catalytic cycle to produce toxic hydroxyl radicals and/or reactiveoxygen species (ROS) (e.g. via the Fenton reaction or Haber Weisscycle). The Fe (III)-trischelate system with redox potential below −320mv (vs NHE or −540 mv vs Ag/AgCl) at pH 7.4 will not be reduced by anybiological reducing agents such as NADPH/NADH, therefore it will notparticipate in the Haber Weiss cycle to generate ROS (reactive oxygenspecies). Within the mammalian body, iron is bound to different proteinssuch as transferrin in human blood to ensure it remains in a form thatcannot react with any oxygen molecules. The E_(1/2) value ofFe-transferrin is −500 mv (vs. NHE or −720 mv vs. Ag/AgCl).

The redox potential of iron complexes can be measured by cyclicvoltammetry (CV). The use of CV to measure the redox potentials of ironchelates deferiprone, deferrioxamine and Apo6619 (a representativecompound of this invention) as chelators respectively, is illustrated inexample 17 below. Iron chelates such as Fe-desferrioxamine (DFO) andFe-(deferiprone)₃ have redox potential E_(1/2) values at −698 mv (vsAg/AgCl) and −834 mv (vs. Ag/AgCl) at pH 7.4 respectively. Compounds offormula I such Fe(Apo6619)₃ has a E_(1/2) value of −691 mv (vs. Ag/AgCl)similar to that of desferrioxamine. The cyclic voltammogram of Fe-DFO,Fe(deferiprone)₃ and Fe(Apo6619)₃ can be found in FIG. 9. One advantageof the chelators of this invention is that the redox potentials of theiriron chelates lie in the extreme negative range at physiological pH 7.4,therefore their iron chelates will not participate in the redox cycle togenerate reactive oxygen species at physiological pH. When combined withother novel properties as described in this invention, the compounds offormula I are effective agents in the removal of iron via a chelationmechanism.

For the treatment of iron overloaded diseases such as thalassemia,sickle cell disease, haemochromatosis and the treatment of patientshaving a toxic concentration of iron, the compounds of the invention maybe administered orally, topically, or parenterally in dosage unitformulations containing conventional non-toxic pharmaceuticallyacceptable carriers, adjuvants and vehicles.

For the treatment of non-iron overloaded conditions such as HIVinfection, protective effect against anthracycline cardiac poisoning,cancer and malaria, the compounds of this invention may also beadministered orally, topically, or parenterally in dosage unitformulations containing conventional non-toxic pharmaceuticallyacceptable carriers, adjuvants and vehicles.

The term parenteral as used herein includes subcutaneous injection orinfusion techniques. In addition to the treatment of warm-bloodedanimals such as mice, rats, horses, cattle, sheep, dogs, cats, etc., thecompounds of the invention are effective in the treatment of humans.

For use in pharmaceutical compositions, conventional non-toxic solidcarriers include, for example, pharmaceutical grades of mannitol,lactose, starch, magnesium stearate, sodium saccharin, talcum,cellulose, glucose, sucrose, magnesium carbonate, and the like may beused. The active compound as defined above may be formulated as aliquid. Pharmaceutically administrable compositions can, for example, beprepared by dissolving, dispersing, etc. an active compound as definedabove and optional pharmaceutically adjuvants in a carrier, such as, forexample, water, saline, aqueous dextrose, glycerol, ethanol, and thelike, to thereby form a solution or suspension. If desired, thepharmaceutical composition to be administered may also contain a minoramount of non-toxic auxiliary substances such as wetting or emulsifyingagents and the like, for example, sodium acetate, sorbitan monolaurate,triethanolamine sodium acetate, triethanol-amine oleate, etc. Actualmethods of preparing such dosage forms are known, or will be apparent tothose skilled in this art: for example, see Remington's PharmaceuticalSciences, Mack Publishing Company, Easton, Pa., 15th Edition, 1975,ch.83 p. 1436-1460, and ch. 89 p. 1576-1607. The composition offormulation to be administered will, in any event, contain a quantity ofthe active compound(s) in an amount effective to alleviate the symptomsof the subject being treated.

The pharmaceutical compositions containing the active ingredient may bein a form suitable for oral use, for example, as tablets, troches,lozenges, aqueous or oily suspensions, dispersible powders or granules,emulsions, hard and soft capsules, or syrups or elixirs. Compositionsintended for oral use may be prepared according to any method known tothe art for the manufacture of pharmaceutical compositions and suchcompositions contain one or more agents from the group consisting ofsweetening agents, flavoring agents, coloring agents and preservingagents in order to provide pharmaceutically elegant and palatablepreparations. Tablets contain the active ingredient in admixture withthe non-toxic pharmaceutically acceptable excipients, which are suitablefor the manufacture of tablets. The excipients may be for example, inertdiluents, such as calcium phosphate or sodium phosphate; granulating anddisintegrating agents, for example, corn starch, or alginic acid;binding agents, for example starch, gelatin or acacia; and lubricatingagents, for example magnesium stearate stearic acid or talc. The tabletsmay be coated by known tecniques to delay the disintegration andabsorption in the gastrointestinal tract and thereby provide a sustainedaction over a long period. For emollient, emulsifier, or moisturer,monostearate or glyceryl distearate may be employed.

Formulations for oral use may also be presented as hard gelatin capsuleswherein the active ingredients are mixed with an inert solid diluent,for example, calcium phosphate or kaolin, or as soft gelatin capsuleswherein the active ingredient is mixed with water or an oil medium, forexample peanut oil, liquid paraffin, or olive oil.

Aqueous suspensions can contain the active materials in an admixturewith the excipient suitable for the manufacture of aqueous suspensions.Such excipients are suspending agents, for example sodiumcarboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose,sodium alginate, polyvinylpyrrolidone, gum and gum acacia; dispersing orwetting agents may be a naturally-occurring phosphate, for examplelecithin, or condensation products of an alkene oxide with fatty acids,for example polyoxyethylene stearate, or condensation products ofethylene oxide with long chain aliphatic alcohols, for exampleheptadecathyl-eneoxycetanol, or condensation products of ethylene oxidewith partial esters derived from fatty acids and hexitol anhydrides, forexample polyethylene sorbitan monooleate. The aqueous suspensions mayalso contain one or more preservatives, for example ethyl, or n-propyl,p-hydroxybenzoate, one or more coloring agents, such as sucrose orsaccharin.

Oily suspensions may be formulated by suspending the active ingredientin a vegetable oil, for example rachis oil, olive oil, sesame oil orcoconut oil, or in a mineral oil such as liquid paraffin. The oilysuspensions may contain a thickening agent, for example beeswax, hardparaffin or cetyl alcohol. Sweetening agents such as those set forthabove, and flavoring agents may be added to provide a palatable oralpreparation. These compositions may be preserved by the addition of ananti-oxidant such as ascorbic acid.

Dispersible powders and granules suitable for preparation of an aqueoussuspension by the addition of water provide the active ingredient inadmixture with the dispersing or wetting agent, suspending agent and oneor more preservatives. Suitable dispersing or wetting agents andsuspending agents are exemplified by those already mentioned above.Additional recipients, for example sweetening, flavoring and coloringagents, may also be present.

The pharmaceutical composition of the invention may also be in the formof oil-in-water emulsions. The oily phase may be a vegetable oil, forexample olive oil or arachis oil, or a mineral oil, for example liquidparaffin or mixtures of these. Suitable emulsifying agents may benaturally occurring phosphates, esters derived from fatty acids andhexitol anhydrides, for example sorbitan monooleate, and condensationproducts of the said partial esters with ethylene oxide, for examplepolyoxyethylene sorbitan monooleate. The emulsion may also containsweetening and flavoring agents.

Syrups and elixirs may be formulated with sweetening agents, for exampleglycerol, propylene glycol, orbital or sucrose. Such formulations mayalso contain a demulcent, a preservative and flavoring and coloringagents. The pharmaceutical compositions may be formulated according tothe known art using those suitable dispersing or wetting agents andsuspending agents, which have been mentioned above. The sterileinjectable preparation may also be a sterile injectable solution orsuspension in a non-toxic parenterally acceptable diluent or solvent,for example as a solution in 1,3-butane diol. Among the acceptablevehicles and solvents that may be employed are water, Ringer's solutionsand isotonic sodium chloride solution. In addition, fixed oils areconventionally employed as a solvent or suspending medium. For thispurpose any bland fixed oil may be employed including synthetic mono- ordiglycerides. In addition, fatty acids such as oleic acid find use inthe preparation or injectables.

Compounds of formula (I), or if appropriate apharmaceutically-acceptable salt thereof and/or apharmaceutically-acceptable solvate thereof, may also be administered asa topical formulation in combination with conventional topicalexcipients. Examples of topical formulations are ointments, creams orlotions, impregnated dressings, gels, gel sticks, spray and aerosols.The formulations may contain appropriate conventional additives such aspreservatives, solvents to assist drug penetration and emollients inointments and creams. They may also contain compatible conventionalcarriers, such as cream or ointment bases and ethanol or oleyl alcoholfor lotions. Topical formulations are envisaged where appropriate, tocontain an amount of actives to alleviate the symptoms of the subjectbeing treated. Suitably, the compound of formula (I), or if appropriatea pharmaceutically-acceptable salt thereof, will compromise from about0.5 to 10% by weight of the formulation. Suitable cream, lotion, gel,stick, ointment, spray or aerosol formulations that may be used forcompounds of formula (I) or if appropriate a pharmaceutically-acceptablesalt thereof, are conventional formulations well known in the art, forexample, as described in standard text books Remington's PharmaceuticalSciences, Mack Publishing Company, Easton, Pa., 15th Edition, 1975, ch.83 p.1436-1460, and ch. 89 p. 1576-1607.

Parenteral administration is generally characterized by injection,either subcutaneously, intramuscularly or intravenously. Injectables canbe prepared in conventional forms, either as liquid solutions orsuspension in liquid prior to injection, or as emulsions. Suitableexcipients are for example, water, saline, dextrose, glycerol, ethanolor the like. In addition, if desired, the pharmaceutical compositions tobe administered may also contain minor amounts of non-toxic auxiliarysubstance such as wetting or emulsifying agents, pH buffering agents andthe like, such as for example, sodium acetate, sorbitan monolaurate,triethanolamine oleate, etc.

The amount of active ingredient that may be combined with the carriermaterials to produce a single dosage form will vary depending upon thehost treated, and the particular mode of administration of humans maycontain from 0.5 mg to 5 gm of active agent compounded with anappropriate and convent amount of carrier material which may vary fromabout 5 to about 95% of the total composition. Dosage unit forms willgenerally contain between from about 1 mg to about 500 mg of an activeingredient.

It will be understood, however, that the specific dose level for anyparticular patient will depend upon a variety of factors including theactivity of the specific compound employed, the age, body weight,general health, sex, diet, time of administration, drug combination andthe severity of the particular disease undergoing therapy.

The compounds of the present invention differ from those compoundsreported in U.S. Pat. Nos. 6,448,273, 6,335,353, RE 35,948 and U.S. Pat.No. 5,688,815. The first three patents describe 3-hydroxy-4-pyridinoneshaving a N1 aliphatic hydrocarbon group. U.S. Pat. No. 5,688,815 alsodescribes 3-hydroxy-4-pyridinones with a N1 substituted or unsubstitutedlower alkyl group. According to a standard chemistry textbook, OrganicChemistry by James B. Hendrickson, Donald J. Cram, George S. Hammond,third edition, 1970, McGraw Hill, p. 72, aliphatic hydrocarbons arecomposed of chains of carbon atoms not arranged in rings. Substancesbelonging to this group are sometimes referred as open chain compounds.Examples of aliphatic hydrocarbon group are linear or branched alkylssuch as methyl, ethyl, propyl, isopropyl, isobutyl, butyl andtert-butyl. The compounds of this invention consist of3-hydroxy-4-pyridinones with (a) N1-cycloalkyl substituent and C2cycloalkylcarbamoyl substituent; or (b) N1-cycloalkyl substituent and C2cycloalkylcarbamoyl substituent; or (c) N1-alkyl substituent with C2cycloalkyl-carbamoyl substituent. They are compounds with acyclichydrocarbon substituents. In acyclic hydrocarbons, the carbon chainsform rings. Examples of acyclic hydrocarbon groups are cycloalkylderivatives such as cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.The four patents, U.S. Pat. Nos. 6,448,273, 6,335,353, RE 35,948 andU.S. Pat. No. 5,688,815 do not cover cycloalkyl derivatives of3-hydroxy-4-pyridinones. This invention covers 3-hydroxy-4-pyridinoneswith a N1-cycloalkyl group with an alkylcarbamoyl group at C2 or acycloalkylcarbamoyl group at C2. It also covers 3-hydroxy-4-pyridinoneswith a cycloalkylcarbamoyl group at C2 with a N1-alkyl group.

The invention is further described and illustrated in the followingspecific examples.

SPECIFIC DESCRIPTION OF THE MOST PREFERRED EMBODIMENTS EXAMPLE 1Preparation of 3-Benzyloxy-6-methyl-4-oxo-4H-pyran-2-carboxylic acidcyclohexylamide

1,1′-carbonyldiimidazole (1.99 g, 12.30 mmol) was added to a solution ofthe 3-(benzyloxy,-6-methyl-4-oxo-4H-pyran-2-carboxylic acid (2.0 g, 7.69mmol) in dimethylformamide (DMF, 18 ml) at room temperature. Theresulting solution was heated at 40°-50° C. for 3 hrs. A light yellowsolution was observed. Cyclohexylamine (1.23 ml, 10.76 mmol) was thenadded. The resulting mixture was stirred at room temperature forovernight. The DMF was removed under reduced pressure to give lightyellow oil as a crude product, which was purified by flash columnchromatography (elution gradient: from 1:1 ethyl acetate/hexane to 10%methanol in ethyl acetate) to yield the titled compound (1.60 g, yield61%) as white solid.

M.p. 118-120° C.; ¹H-NMR (CDCl₃, 400 MHz) δ 0.91 (m, 2H, cyclohexyl-H),1.29 (m, 2H, cyclohexyl-H), 1.58 (m, 3H, cyclohexyl-H), 1.79 (m, 2H,cyclohexyl-H), 2.37(s, 3H, CH₃), 3.79 (m, 1H, CH), 5.40(s, 2H, CH₂),6.28(s, 1H, CH), 7.41(m, 5H, ArH), 7.67 (br, 1H, NH); MS (m/z) 342(M⁺+1).

In a similar manner to that described above, by substitutingcyclohexylamine with other amine, the following compounds are prepared:

3-Benzyloxy-6-methyl-4-oxo-4H-pyran-2-carboxylic acid cyclopropylamide

M.p. 79-80° C.; ¹H-NMR (CDCl₃, 400 MHz) δ 0.21 (m, 2H, cyclopropyl-H),0.70 (m, 2H, cyclopropyl-H), 2.35 (s, 3H, CH₃), 2.71 (m, 1H, CH), 5.35(s, 2H, CH₂), 6.27 (s, 1H, CH), 7.39 (m, 5H, ArH), 7.70 (s, 1H, NH);¹³C(CDCl₃) δ 6.35, 7.21, 19.87, 22.61, 22.70, 75.56, 115.37, 128.94(2C),129.17(2C), 129.25, 135.49, 146.14, 146.39, 160.22, 165.74, 176.17; MS(m/z) 300 (M⁺+1).

3-Benzyloxy-6-methyl-4-oxo-4H-pyran-2-carboxylic acid methylamide

M.p. 137-140° C., ¹H-NMR (CDCl₃, 400 MHz) δ 2.38 (s, 3H, CH₃), 2.78 (d,3H, NCH₃), 5.39 (s, 2H, CH₂), 6.30 (s, 1H, CH), 7.40 (m, 5H, ArH), 7.62(br, 1H, NH); MS (m/z) 300 (M⁺+1).

EXAMPLE 2 Preparation of3-Benzyloxy-1,6-dimethyl-4-oxo-1,4-dihydro-pyridine-2-carboxylic acidcyclohexylamide

To a solution of 3-benzyloxy-6-methyl-4-oxo-4H-pyran-2-carboxylic acidcyclohexylamide (1.40 g, 4.1 mmol) in 5 ml of methanol, methylaminesolution (9 ml of 2M solution in methanol, 16 mmol) was added. Theresulting solution was stirred at 70 to 75° C. for overnight under thepressure in a sealed tube. The solvent was removed under reducedpressure gave light yellow solid as a crude product. The material waspurified by column chromatography (elution gradient: 100% ethyl acetateto 25% methanol in ethyl acetate) to give the titled compound as whitesolid (1.20 g, 83.0%).

M.p. 258-260° C.; ¹H-NMR (CDCl₃, 400 MHz) δ 1.26-1.45 (m, 6H,cyclohexyl-H), 1.79 (m, 2H, cyclohexyl-H), 1.95(m, 2H, cyclohexyl-H),2.41(s, 3H, CH₃), 3.82(s, 3H, NCH₃), 3.95 (m, 1H, CH), 5.13 (s, 2H,CH₂), 7.19 (s, 1H, CH), 7.36 (m, 3H, ArH), 7.43(m, 2H, ArH), 8.50 (br,1H, NH); MS (m/z) 355 (M⁺+1).

In a similar manner, by substituting3-benzyloxy-6-methyl-4-oxo-4H-pyran-2-carboxylic acid cyclohexylamidewith other 3-benzyloxy-6-methyl-4-oxo-4H-pyran-2-carboxylic acid amidederivatives, the following compounds are prepared:

3-Benzyloxy-1,6-dimethyl-4-oxo-1,4-dihydro-pyridine-2-carboxylic acidcyclopropylamide

M.p. 187-189° C.; ¹H-NMR (CDCl₃, 400 MHz) δ 0.52 (m, 2H, cyclopropyl-H),0.74 (m, 2H, cyclopropyl-H), 2.18 (s, 3H, CH₃), 2.78 (m, 1H, CH), 3.50(s, 3H, NCH₃), 5.08 (s, 2H, CH₂), 6.12 (s, 1H, CH), 7.33 (m, 3H, ArH),7.39(m, 2H, ArH), 7.91 (br, 1H, NH); MS (m/z) 313 (M⁺+1).

3-Benzyloxy-1-cyclopropyl-6-methyl-4-oxo-1,4-dihydro-pyridine-2-carboxylicacid methylamide

M.p. 132-135° C.; ¹H-NMR (CDCl₃, 400 MHz) δ 1.05 (m, 4H, cyclopropyl-H),2.38 (s, 3H, CH₃), 2.70 (d, J=1.8 Hz, 3H, NCH₃), 3.35 (m, 1H, CH), 5.07(s, 2H, CH₂), 6.14(s, 1H, CH), 7.15 (br., 1H), 7.35 (m, 5H, ArH);¹³C(CDCl₃) δ 9.48, 20.30, 25.86, 34.15, 74.01, 118.16, 127.79,128.06(2C), 128.22(2C), 137.35, 142.05, 143.98, 149.91, 162.01, 173.89;MS (m/z) 313 (M⁺+1).

3-Benzyloxy-1-cyclopropyl-6-methyl-4-oxo-1,4-dihydro-pyridine-2-carboxylicacid cyclopropylamide

M.p. 164-167° C.; ¹H-NMR (CDCl₃, 400 MHz) δ 0.54 (m, 2H, cyclopropyl-H),0.76 (m, 2H, cyclopropyl-H), 1.08-1.11 (m, 4H, cyclopropyl-H), 2.35 (s,3H, CH₃), 2.75 (m, 1H, CH), 3.37 (m, 1H, CH), 5.05 (s, 2H, CH₂), 6.13(s, 1H, CH), 7.33 (m, 5H, ArH), 7.89 (br, s, 1H, NH); MS (m/z) 339(M⁺+1).

EXAMPLE 3 3-Hydroxy-1,6-dimethyl-4-oxo-1,4-dihydro-pyridine-2-carboxylicacid cyclohexylamide [Apo6621]

Pd(OH)₂ on charcoal (0.18 g, 10% w dry basis) was added to a solution of3-benzyloxy-1,6-dimethyl-4-oxo-1,4-dihydro-pyridine-2-carboxylic acidcyclohexylamide (1.0 g, 2.82 mmol) in ethanol (50 ml) under nitrogen.The mixture was hydrogenated at 50 psi for 4 hrs. The Pd(OH)₂ wasremoved by filtration through a layer of Celite, the Celite cake wasthen washed with ethanol (3×10 ml). The ethanol filtrate was evaporatedto give an off-white solid (0.57 g, 77%). Further purification byrecrystallization from methanol (15 ml) gave the title compound as awhite solid (0.18 g). M.p. 280-285° C. (dec); ¹H-NMR (CD₃OD 400 MHz) δ1.30-1.43 (m, 5H, cyclohexyl-H), 1.70 (m, 1H, cyclohexyl-H), 1.80 (m,2H, cyclohexyl-H), 2.00 (m, 2H, cyclohexyl-H), 2.41 (s, 3H, CH₃), 3.63(s, 3H, CH₃), 3.90 (m, 1H, CH), 6.38 (s, 1H, CH); MS (m/z) 265 (M⁺+1).

In a similar manner, by substituting3-benzyloxy-1,6-dimethyl-4-oxo-1,4-dihydro-pyridine-2-carboxylic acidcyclohexylamide with other3-benzyloxy-4-oxo-1,4-dihydro-pyridine-2-carboxylic acid cycloalkylamides, the following compounds are prepared:

3-Hydroxy-1,6-dimethyl-4-oxo-1,4-dihydro-pyridine-2-carboxylic acidcyclopropylamide [Apo6617]

M.p. 260-262° C.; ¹H-NMR (MeOD-d₄, 400 MHz) δ 0.66 (m, 2H,cyclopropyl-H), 0.85 (m, 2H, cyclopropyl-H), 2.41 (s, 3H, CH₃), 2.95 (m,1H, CH), 3.63 (m, 1H, NCH₃), 6.38(s, 1H, CH); MS (m/z) 223 (M⁺+1).

1-cyclopropyl-3-hydroxy-6-methyl-4-oxo-1,4-dihydro-pyridine-2-carboxylicacid methylamide [Apo6619]

M.p. 258-260° C. (dec); ¹H-NMR (MeOD-d₄, 400 MHz) δ1.05 (m, 2H,cyclopropyl-H), 1.19 (m, 2H, cyclopropyl-H), 2.54 (s, 3H, CH₃), 2.97 (s,1H, NCH₃), 3.46 (m, 1H, CH), 6.33 (s, 1H, CH); MS (m/z) 223 (M⁺+1).

EXAMPLE 4 Preparation of3-(benzyloxy)-N-cyclobutyl-6-methyl-4-oxo-4H-pyran-2-carboxamide

A mixture of 3-(benzyloxy)-6-methyl-4-oxo-4H-pyran-2-carboxylic acid(2.5 g, 9.6 mmol, 1.0 equiv), 1,1′-carbonyldiimidazole (2.49 g, 15.37mmol, 1.6 equiv) in DMF (20 mL) was stirred at 50° C. for 5 h. Themixture was cooled to room temperature. Cyclobutylamine hydrochloride(1.24 g, 11.52 mmol, 1.2 equiv) and Et₃N (1.74 mL, 12.48 mmol, 1.3equiv) was added, and the mixture was stirred overnight at roomtemperature. The solvent was removed under reduced pressure.Purification by chromatography (1:1 hexanes/EtOAc, then EtOAc) providedthe titled compound (2.76 g, 91.56%) as a yellow solid.

M.p. 69.3-71.0° C.; ¹H-NMR (CDCl₃, 400 MHz) δ 1.51-1.72 (m, 4H,cyclobutyl H), 2.19-2.28 (m, 2H, cyclobutyl H), 2.37 (s, 3H, CH₃),4.39-4.41 (m, 1H, CH), 5.41 (s, 2H, OCH₂Ph), 6.30 (s, 1H, CH), 7.39-7.49(m, 5H, ArH), 7.86 (br, 1H, NH), and MS (m/z) 314 (M⁺+1), 217, 91.

Proceeding in a similar manner, the following compound is prepared:

3-(benzyloxy)-N-cyclopentyl-6-methyl-4-oxo-4H-pyran-2-carboxamide

M.p. 108.0-108.5° C.; ¹H-NMR (CDCl₃, 400 MHz) δ1.11-1.16 (m, 2H,cyclopentyl H), 1.50-1.55 (m, 4H, cyclopentyl H), 1.87-1.92 (m, 2H,cyclopentyl H), 2.38 (s, 3H, CH₃), 4.17-4.22 (m, 1H, CH), 5.41 (s, 2H,CH₂), 6.30 (s, 1H, CH), 7.38-7.43 (m, 5H, ArH), 7.72 (br, 1H, NH), MS(m/z) 328(M⁺+1), 217, 91.

EXAMPLE 5 Preparation of3-(benzyloxy)-N-cyclobutyl-1,6-dimethyl-4-oxo-1,4-dihydropyridine-2-carboxamide

To a solution of the compound from example 4 (2.616 g, 8.35 mmol, 1.0equiv) in methanol (10 ml) was quickly added methylamine (2M inmethanol, 20 ml, 40 mmol, 4.79 equiv). The sealed tube was stirredovernight at 70-75° C. The resulting brown solution was evaporated todryness and purified by chromatography (EtOAc, then 1:4 MeOH/EtOAc)provided the titled compound (1.70 g, 62.24%) as a white solid.

M.p. 221.3-222.4° C.; ¹H-NMR (DMSO-d₆, 400 MHz) δ 1.65-1.69 (m, 2H,cyclobutyl H), 1.90-1.95 (m, 2H, cyclobutyl H), 2.14-2.21 (m, 2H,cyclobutyl H), 2.31 (s, 3H, CH₃), 3.42 (s, 3H, NCH₃), 4.34-4.30 (m, 1H,CH), 5.05 (s, 2H, OCH₂Ph), 6.22 (s, 1H, CH), 7.39-7.30 (m, 5H, ArH),9.08-9.06 (d, 1H, J=7.08 Hz, NH); MS (m/z) 327(M⁺+1), 230, 166, 91.

Proceeding in a similar manner, the following compound is prepared:

3-(benzyloxy)-N-cyclopentyl-1,6-dimethyl-4-oxo-1,4-dihydropyridine-2-carboxamide

M.p. 233.6-234.4° C.; ¹H-NMR (DMSO-d₆, 400 MHz) δ 1.43-1.52 (m, 4H,cyclopentyl H), 1.54-1.60 (m, 2H, cyclopentyl H), 1.78-1.83 (m, 2H,cyclopentyl H), 2.30 (s, 3H, CH₃), 3.43 (s, 3H, NCH₃), 4.13-4.18 (m, 1H,CH), 5.04 (s, 2H, OCH₂Ph), 6.22 (s, 1H, CH), 7.30-7.41 (m, 5H, ArH),8.80-8.82 (d, J=6.95 Hz, 1H, NH); MS (m/z) 341(M⁺+1), 230, 166, 91.

EXAMPLE 6 Preparation ofN-cyclobutyl-3-hydroxy-1,6-dimethyl-4-oxo-1,4-dihydropyridine-2-carboxamide[Apo6622]

A mixture ofN-cyclobutyl-3-benzyloxy-1,6-dimethyl-4-oxo-1,4-dihydropyridine-2-carboxamide(1.528 g, 4.68 mmol, 1.0 equiv), 10% Pd on activated carbon (200 mg,wet), and ethanol (200 ml) was stirred under 50 psi of H₂ at roomtemperature for 2.5 h. The catalyst was filtered through Celite and thefiltrate was evaporated to give a solid, which was recrystallized fromMeOH gave the titled compound (0.57 g, 51.5%) as a white solid.

M.p. 277.3° C. (dec); ¹H-NMR (DMSO-d₆, 400 MHz) δ 1.68-1.70 (m, 2H,cyclobutyl H), 1.95-2.01 (m, 2H, cyclobutyl H), 2.20-2.26 (m, 2H,cyclobutyl H), 2.29 (s, 3H, CH₃), 3.41 (s, 3H, NCH₃), 4.31-4.35 (m, 1H,CH), 6.13 (s, 1H, CH), 8.98 (br, 1H, NH); MS (m/z) 237 (M⁺+1), 185, 166,123.

Proceeding in a similar manner, the following compounds are prepared:

N-cyclopentyl-3-hydroxy-1,6-dimethyl-4-oxo-1,4-dihydro-pyridine-2-carboxamide[Apo6620]

M.p. 289.3° C. (dec); ¹H-NMR (DMSO-d₆, 400 MHz) δ 1.49-1.55 (m, 4H,cyclopentyl H), 1.61-1.68 (m, 2H, cyclopentyl H), 1.83-1.87 (m, 2H,cyclopentyl H), 2.29 (s, 3H, CH₃), 3.42 (s, 3H, NCH₃), 4.14-4.18 (m, 1H,CH), 6.12 (s, 1H), 8.71-8.73 (d, J=7.05 Hz, 1H, NH); MS (m/z) 251(M⁺+1), 166.

1-Cyclopropyl-3-hydroxy-6-methyl-4-oxo-1,4-dihydro-pyridine-2-carboxylicacid cyclopropylamide [Apo6618].

M.p. 241-143° C.; ¹H-NMR (DMSO-d₆, 400 MHz) δ 0.53 (m, 2H,cyclopropyl-H), 0.71 (m, 2H, cyclopropyl-H), 0.94-1.00 (m, 4H,cyclopropyl-H), 2.42 (s, 3H, CH₃), 2.79 (m, 1H, CH), 3.30 (m, 1H, CH),6.08 (s, 1H, CH), 8.54 (br, s, 1H, NH); MS (m/z) 249 (M⁺+1).

EXAMPLE 7 Preparation of3-benzyloxy-1-cyclopropyl-6-methyl-oxo-1,4-dihydro-pyridine-2-carboxylicacid

To a suspension of 3-(benzyloxy)-6-methyl-4-oxo-4H-pyran-2-carboxylicacid (70 g, 0.27 mol) in MeOH (350 mL) in a 3-necked RBF (round bottomflask) fitted with a mechanical stirrer was added cyclopropylamine (120mL, 1.72 mol). A clear light yellow solution resulted. The reactionmixture was refluxed for ca. 19 h. Volatiles were removed in vacuo andthe residue was dissolved in water (700 mL) with stirring. The aqueousmixture was filtered through a pad of Celite®. The filtrate was placedin a 3-necked RBF fitted with a mechanical stirrer, and cooled in an icebath. Conc. HCl was added until the pH was ca. 1-2, and voluminous“orange” solid precipitated out. Acetone (200 mL) was added to thesuspension. The solid was then collected by suction filtration,thoroughly washed with acetone, and air-dried. The title compound wasobtained as an off-white solid (71.0 g, 88%).

Mp: 139.0-139.5° C.; ¹H-NMR (300 MHz, DMSO-D₆) δ (ppm): 0.98-1.15 (m,4H, 2 c-CH₂), [2.37 (s)+2.40 (s), rotamers, 3/2 ratio, 3H, CH₃)],3.30-3.50 (m, 1H, c-CH), 5.00-5.05 (m, 2H, CH₂Ph), 6.20-6.25 (m, 1H,C=CH), 7.28-7.50 (m, 5H, Ph); MS (m/z): 300.2 (M⁺+1), 256.2, 192.2,164.4, 91.0 (100%);

Anal. Calcd. for C₁₇H₁₇NO₄: C, 68.21; H, 5.72; N, 4.68%. Found: C,67.76; H, 5.76; N, 4.61%.

EXAMPLE 8 Synthesis of3-benzyloxy-1-cyclopropyl-6-methyl-4-oxo-1,4-dihydro-pyridine-2-carboxylicacid methylamide

To a cold suspension (ice-salt bath, internal temp=−5° C.) of3-benzyloxy-1-cyclopropyl-6-methyl-oxo-1,4-dihydro-pyridine-2-carboxylicacid (30 g, 0.10 mol), CH₂Cl₂ (150 mL) and DMF (7.8 mL, 0.10 mol) in a3N-RBF (round bottom flask) fitted with a mechanical stirrer was addedthionyl chloride (9.5 mL, 0.13 mol) dropwise over a period of 5 minutes.After addition of thionyl chloride, the reaction mixture was still asuspension. The ice-salt bath was removed. The reaction mixture wasallowed to warm up to room temperature. Aliquots were removed andquenched with a 2M methylamine solution in THF. The resulting mixturewas then analyzed by HPLC. Thus, HPLC monitoring indicated about 96%consumption of starting material after the reaction mixture was stirredat room temperature for 3 h (HPLC, mobile phase: 0.035% HClO₄/CH₃CN,80/20, column: symmetry C18 WAT046980, flow rate: 1 ml/min, monitoringwavelength: 260 nm, RT of3-benzyloxy-1-cyclopropyl-6-methyl-oxo-1,4-dihydro-pyridine-2-carboxylicacid=2.46 min, RT of3-benzyloxy-1-cyclopropyl-6-methyl-4-oxo-1,4-dihydro-pyridine-2-carboxylicacid methylamide=5.40 min).

In another 1-L 3N-RBF fitted with a mechanical stirrer was placeddichloromethane (240 mL) and triethylamine (36 mL, 0.26 mol) (ice-saltbath, internal temp=−10° C.). 2M methylamine in tetrahydrofuran (73 mL,0.146 mol) was added to the cold solution. The acid chloride generatedin situ above was transferred to an addition funnel, and slowly added tothe amine solution over a period of 30 minutes. An exothermic reactionwas noticed, but the internal T was kept at below −5° C. The reactionwas completed after 10 min as indicated by TLC (CH₂Cl₂/MeOH, 9/1 ratio,v/v). The reaction mixture was quenched with water (100 mL), and themixture was stirred for 5 min. The organic fraction was collected andwashed twice more with water, followed by washing with diluted NaOHsolution (0.05 M, 3×100 mL), dried over sodium sulfate, filtered andconcentrated in vacuo to afford a brown solid. The solid was suspendedin 150 mL of a mixture of ethanol and ethyl acetate (2/8 ratio, v/v),and the slurry was stirred for 2 h. The solid was collected by suctionfiltration, washed with ethyl acetate (50 mL), and was then air-dried.The title compound was thus obtained as a light-pink, slightly brownishsolid (14 g, 45%). The material was further purified by columnchromatrography (5% MeOH:CH₂Cl₂).

M.p. 132-135° C.; ¹H-NMR (CDCl₃, 400 MHz) δ 1.05 (m, 4H, cyclopropyl-H),2.38 (s, 3H, CH₃), 2.70 (d, J=1.8 Hz, 3H, NCH₃), 3.35 (m, 1H, CH), 5.07(s, 2H, CH₂), 6.14(s, 1H, CH), 7.15 (br, 1H), 7.35 (m, 5H, ArH);¹³C(CDCl₃) δ 9.48, 20.30, 25.86, 34.15, 74.01, 118.16, 127.79,128.06(2C), 128.22(2C), 137.35, 142.05, 143.98, 149.91, 162.01, 173.89;MS (m/z): 313 (M⁺+1).

In a similar manner, the following compounds were prepared:

3-Benzyloxy)-N-(cyclohexylmethyl)-1-cyclopropyl-6-methyl-4-oxo-1,4-dihydropyridine-2-carboxamide

¹H-NMR (CD₃OD, 400 MHz) δ 0.90-0.96 (m, 3H), 1.13-1.23 (m, 3H),1.45-1.54 (m, 1H), 1.64 (br.m, 4H), 1.73-1.76 (br.m, 4H), 2.56 (s, 3H,CH₃), 3.12-3.13 (d, J=6.8 Hz, 2H), 3.36-3.40 (m, 1H, CH), 5.09 (s, 2H),6.43 (s, 1H), 7.31-7.37 (m, 3H), 7.43-7.45 (m, 2H); MS (m/z): 395(M⁺+1).

3-(Benzyloxy)-1-cyclopropyl-6-methyl-2-(morpholin-4-ylcarbonyl)pyridin-4(1H)-one

¹H-NMR (CDCl₃, 400 MHz) δ 0.87-0.94 (br.m, 1H), 1.09-1.13 (m, 1H),1.25-1.30 (m, 2H), 2.56 (s, 3H, CH₃), 3.30-3.42 (m, 2H), 3.45-3.69 (m,6H), 3.84-3.90 (m, 1H, CH), 4.74-4.77 (d, J=10.4 Hz, 1H), 5.54-5.56 (d,J=10.6 Hz, 1H), 6.80 (br.s, 1H, NH), 7.36-7.41 (m, 5H, ArH); MS (m/z):369 (M⁺+1). cl3-(Benzyloxy)-1-cyclopropyl-6-methyl-N-(3-methylbutyl)-4-oxo-1,4-dihydropyridine-2-carboxamide

¹H-NMR (CDCl₃, 400 MHz) δ 0.86-0.88 (d, J=6.4 Hz, 6H, 2CH₃), 1.04-1.09(m, 4H), 1.27-1.37 (m, 2H), 1.55-1.60 (m, 1H, CH), 2.37 (s, 3H, CH₃),3.20-3.25 (m, 2H, CH₂), 3.34-3.37 (m, 1H, CH), 5.09 (s, 2H, CH₂), 6.10(s, 1H), 7.30-7.38 (m, 5H, ArH), 7.23-2.28 (br.t, 1H, NH).

3-(Benzyloxy)-N-cyclohexyl-1-cyclopropyl-6-methyl-4-oxo-1,4-dihydropyridine-2-carboxamide

¹H-NMR (CDCl₃, 400 MHz) δ 1.15-1.30 (m, 3H), 1.31 (br.m, 1H), 1.34(br.m, 5H), 1.66-1.70 (m, 1H), 2.78 (s, 3H, CH₃), 3.30-3.34 (m, 1H),3.42-3.51 (m, 2H), 3.67-3.69 (m, 1H), 3.80-3.83 (m, 1H), 4.82-4.85 (d,J=10.3 Hz, 1H), 5.37-5.40 (d, J=10.5 Hz, 1H), 7.34 (br.m, 5H, ArH), 7.86(s, 1H).

3-(Benzyloxy)-1-cyclopropyl-N-hexyl-6-methyl-4-oxo-1,4-dihydropyridine-2-carboxamide

¹H-NMR (CDCl₃, 400 MHz) δ 0.89-0.92 (t, J=6.6 Hz, 3H, CH₃), 1.25-1.32(m, 6H), 1.40-1.47 (m, 4H), 1.64-1.70 (m, 2H, CH₂), 2.54 (s, 3H, CH₃),3.43-3.48 (m, 2H, CH₂), 3.91-3.93 (m, 1H, CH), 5.10 (s, 2H, CH₂),7.37-7.46 (m, 6H, ArH and C═CH), 9.24 (br.t, 1H, NH); MS (m/z): 383(M⁺+1).

3-(Benzyloxy)-1-cyclopropyl-6-methyl-2-[(4-methylpiperazin-1-yl)carbonyl]pyridin-4(1H)-one

¹H-NMR (CDCl₃, 400 MHz) δ 0.85-0.88 (m, 1H), 1.06-1.29 (m, 4H),1.40-1.45 (br.m, 2H), 1.50-1.58 (br.m, 4H), 2.51 (s, 3H, CH₃), 3.12-3.17(m, 1H), 3.35-3.48 (m, 3H), 3.75-3.78 (m, 1H, CH), 4.76-4.78 (d, J=10.6Hz, 1H), 5.53-5.56 (d, J=10.7 Hz, 1H), 6.68 (br.s, 1H, NH), 7.30-7.43(m, 5H, ArH); MS (m/z): 382 (M⁺+1).

3-(Benzyloxy)-1-cyclopropyl-N,N,6-trimethyl-4-oxo-1,4-dihydropyridine-2-carboxamide

¹H-NMR (CDCl₃, 400 MHz) δ 1.16-1.20 (m, 2H), 1.27-1.33 (m, 1H),1.87-1.95 (m, 1H), 2.78 (s, 3H, CH₃), 3.05 (s, 3H, CH₃), 3.08 (s, 3H,CH₃), 3.62-3.68 (m, 1H, CH), 4.86-4.90 (d, J=10.8 Hz, 1H), 5.33-5.38 (d,J=10.8 Hz, 1H), 7.29-7.33 (m, 5H, ArH), 7.77 (s, 1H, NH); MS (m/z): 327(M⁺+1).

EXAMPLE 9 A. Preparation of1-cyclopropyl-3-hydroxy-6-methyl-4-oxo-1,4-dihydro-pyridine-2-carboxylicacid methylamide

Procedure I:

Step a. Synthesis of1-cyclopropyl-3-hydroxy-6-methyl4-oxo-1,4-dihydro-pyridine-2-carboxylicacid methylamide.

To a suspension of3-benzyloxy-1-cyclopropyl-6-methyl4-oxo-1,4-dihydro-pyridine-2-carboxylicacid methylamide (10.0 g, 0.032 mol) in methanol (40 mL) and water (2.6mL) at ice-bath temperature, was added conc. HCl (3.9 mL) dropwise. Theresulting clear brown solution was stirred at room temperature for ca. 5min, then nitrogen gas was bubbled into the solution for ca. 5 min. Pd-C(10% wet, 5%w/w, 0.5 g) was added and the reaction vessel was purgedwith hydrogen twice. The mixture was hydrogenated in a Parr reactorunder 50 psi hydrogen pressure at RT, and the progress of the reactionwas monitored by HPLC over 3 h. The reaction was over after 3h.

Excess hydrogen was evacuated and nitrogen gas was bubbled into thesolution for about 5 min. The reaction mixture was filtered overpre-treated celite (previously washed with a 0.1N standard solution of1-cyclopropyl-3-hydroxy-6-methyl-4-oxo-1,4-dihydro-pyridine-2-carboxylicacid methylamide in methanol), and the cake was washed with 6×10 mL ofmethanol. The volume of the filtrate was reduced to about 30 mL underreduced pressure. The residue was cooled in ice and some solid startedto precipitate out. A 2N NaOH solution (25 mL) was added until the pHwas about 5, and the mixture was stirred for about 10 min. Methyltert-butyl ether (MTBE, 30 mL) was added, and the resulting mixture wasstirred at ice-bath temperature for 30 min. The solid was collected bysuction filtration, twice thoroughly washed with a mixture of 5 mL ofEtOH/MTBE (1/2 ratio). HPLC condition for reaction monitoring usingHewlett Packard series 1100HPLC: symmetry C18 column (WAT046980),gradient 0.035% HClO₄/CH₃CN, min-% CH₃CN: 0-10; 6-10; 7-20 and 15-20, λat 210, 260 and 285 nm; retention time of1-cyclopropyl-3-hydroxy-6-methyl-4-oxo-1,4-dihydro-pyridine-2-carboxylicacid methylamide is 2.099 min.

Step b. Purification of1-cyclopropyl-3-hydroxy-6-methyl-4-oxo-1,4-dihydro-pyridine-2-carboxylicacid methylamide.

The suspension of crude product obtained as described in Step a in a 1/1mixture of EtOH/distilled water (14 mL total) was stirred at ice-bathtemperature for 1 h. The solid was collected by suction filtration, andwashed 2× thoroughly with 5 mL of a 1/1 mixture of pre-cooledEtOH/distilled water. The title compound, a light pinkish solid, wasdried to constant weight at 40° C. under vacuum for 16 h. This productgave a negative silver nitrate test, and weighed 5.3 g (74% total yield,steps a and b).

¹H-NMR (300 MHz, DMSO-D₆) δ (ppm): 0.94-0.99 (m, 4H, 2 c-CH₂), 2.39 (s,3H, CCH₃), 2.76 (d, J=4.4 Hz, 3H, NHCH₃), 3.28-3.31 (m, 1H, c-CH), 6.08(s, 1H, C═CH), 8.44 (br. q., 1H, NHCH₃);¹³C-NMR (75 MHz, DMSO-D₆) δ(ppm): 9.1, 19.9, 25.8, 33.7, 112.3, 130.1, 143.3, 148.7, 161.8, 170.6;MS/MS (+ve ES): MS (m/z) 223 (M⁺+1), 192.1, 164.2 (M⁺− CONHCH₃, 100%),150.1, 136.3; Elemental Analysis:

Anal. Calcd. for C₁₁H₁₄N₂O₃: C, 59.45; H, 6.35; N, 12.60%. Found: C,59.19; H, 6.07; N, 12.53%; IR (KBr) cm⁻¹: 3300 (NH), 1670, 1653, 1495(C═C).

B.N-(Cyclohexylmethyl)-1-cyclopropyl-3-hydroxy-6-methyl-4-oxo-1,4-dihydropyridine-2-carboxamide:

A mixture of3-(benzyloxy)-N-(cyclohexylmethyl)-1-cyclopropyl-6-methyl-4-oxo-1,4-dihydropyridine-2-carboxamide(2.0 g, 4.8 mmol), Pd/C (10% wet, 0.45 g) in ethanol (150 mL) washydrogenated in a Parr apparatus at 50 psi of hydrogen pressure for 16h. The reaction mixture was filtered over a pad of celite and the celitewas thoroughly washed with EtOH (25 mL). Evaporation of the solventafforded a pale pink solid. The solid was dissolved in hot methanol,then cooled to RT as solid product precipitated out. The solid wascollected by suction filtration. The mother liquor was concentrated invacuo and the residual solid was again dissolved in hot methanol andcooled to RT to precipitate out the product, which was then collected.This process was repeated one more time. The three combined white solidfractions weighed 0.95 g (63% yield).

¹H-NMR (CDCl₃, 400 MHz) δ 0.84-0.88 (m, 2H, CH₂ of c-Pr), 1.03-1.09 (m,2H, CH₂ of c-Pr)), 1.06-1.31 (m, 5H), 1.65-1.87 (m, 6H), 2.50 (s, 3H,CH₃), 3.33-3.36 (m, 2H, CH₂N), 3.51 (s, 1H), 3.58-3.61 (m, 1H, CH ofc-Pr), 6.27 (s, 1H, C═CH), 6.80 (br.t, 1H, NH); MS (m/z): 305 (M⁺+1).

C. The Following Compounds were Prepared in a Similar Fashion:

1-Cyclopropyl-3-hydroxy-6-methyl-N-(3-methylbutyl)4-oxo-1,4-dihydropyridine-2-carboxamide

Yield: 88%; ¹H-NMR (CDCl₃, 400 MHz) δ 0.85-0.89 (m, 1H), 0.98-1.00 (d,J=6.4 Hz, 6H, 2CH₃), 1.15-1.19 (m, 2H), 1.54-1.60 (m, 2H), 1.72-1.77 (m,1H, CH), 2.50 (s, 3H, CH₃), 3.49-3.53 (m, 2H, CH₂), 3.57-3.60 (m, 1H,CH), 3.72 (br.s, 1H), 6.27 (s, 1H), 7.23 (br.t, 1H, NH); MS (m/z): 279(M⁺+1).

1-Cyclopropyl-N-hexyl-3-hydroxy-6-methyl-4-oxo-1,4-dihydropyridine-2-carboxamide

Yield: 87%; ¹H-NMR (CDCl₃, 400 MHz) δ 0.90-0.94 (t, J=6.8 Hz, 3H, CH₃),1.27-1.47 (m, 10H), 1.68-1.73 (m, 2H), 2.70 (s, 3H, CH₃), 3.47-3.52 (m,2H, CH₂), 3.85-3.88 (m, 1H, CH), 7.05 (s, 1H, C═CH), 8.30 (br.t, 1H,NH); MS (m/z): 293 (M⁺+1).

N-Cyclohexyl-1-cyclopropyl-3-hydroxy-6-methyl-4-oxo-1,4-dihydropyridine-2-carboxamide

Yield: 91%; ¹H-NMR (CDCl₃, 400 MHz) δ 0.98-1.05 (m, 1H), 1.21-1.38 (m,3H), 1.60-1.80 (br.m, 7H), 2.71 (s, 3H, CH₃), 3.32-3.37 (m, 1H),3.46-3.50 (m, 1H), 3.55-3.64 (m, 2H), 3.92-3.99 (m, 1H), 6.88 (s, 1H,C═CH); MS (m/z): 277 (M⁺+1).

1-Cyclopropyl-3-hydroxy-N,N,6-trimethyl-4-oxo-1,4-dihydropyridine-2-carboxamide

Yield: 97%; ¹H-NMR (CD₃OD, 300 MHz) δ 0.98-1.10 (m, 1H), 1.15-1.43 (m,3H), 2.76 (s, 3H, CH₃), 3.07 (s, 3H, CH₃), 3.16 (s, 3H, CH₃), 3.70-3.76(m, 1H, CH), 7.10 (s, 1H, C═CH); ¹³C-MR (CD₃OD, 75 MHz) δ 9.5, 10.9,21.3, 35.0, 38.1, 38.8, 114.4, 138.8, 142.9, 154.7, 162.5, 162.8; MS(m/z): 237 (M⁺+1).

1-Cyclopropyl-3-hydroxy-6-methyl-2-1[(4-methylpiperazin-1-yl)carbonyl]pyrdin-4(1H)-one

Yield: 96%; ¹H-NMR (CD₃OD, 300 MHz) δ 0.89-1.00 (m, 1H), 1.06-1.29 (m,3H), 1.52-1.85 (br.m, 8H), 2.56 (s, 3H, CH₃), 3.40-3.60 (m, 3H),3.88-3.98 (m, 1H, CH), 6.48 (s, 1H, C═CH); ¹³C-NMR (CD₃OD, 75 MHz) δ10.0, 11.0, 21.0, 25.4, 26.4, 27.0, 36.5, 43.8, 49.2, 114.7, 132.9,144.5, 152.8, 162.4, 170.2.

N,1-Dicyclopropyl-3-hydroxy-6-methyl-4-oxo-1,4-dihydropyridine-2-carboxamide

¹H-NMR (CDCl₃, 400 MHz) δ 0.68-0.70 (m, 2H), 0.85-0.95 (m, 4H),1.15-1.26 (m, 2H), 2.70 (s, 3H, CH₃), 2.91-2.98 (m, 1H), 3.50-3.61 (m,1H), 6.26 (s, 1H, C═CH), 7.10 (br.s, 1H, NH); MS (m/z): 249 (M⁺+1).

1-Cyclopropyl-3-hydroxy-6-methyl-2-(morpholin-4-ylcarbonyl)pyridin-4(1H)-one

¹H-NMR (CD₃OD, 300 MHz) δ 1.00-1.10 (m, 1H), 1.20-1.45 (m, 3H), 2.73 (s,3H, CH₃), 3.45-3.53 (m, 2H), 3.62-3.86 (m, 6H), 3.90-4.00 (m, 1H), 7.02(s, 1H, C═CH); ¹³C-NMR (CD₃OD, 75 MHz) δ 10.3, 11.1, 21.3, 38.6, 43.6,48.3, 67.4, 67.7, 114.5, 137.2, 143.3, 154.7, 161.2, 163.7; MS (m/z):279 (M⁺+1).

EXAMPLE 10 pKa Determination for Apo6619 by Potentiometric Titration

The pKa values of ligands were determined by potentiometric titrationwhen a ligand concentration greater than 1×10⁻² M in water could beprepared. In a typical experiment, the sample solution (2.67×10⁻² M) wasprepared by the following method: Apo6619 (92.6 mg) was weighed into a25-ml beaker, followed by the addition of 0.1 M NaCl (15 ml). Themixture was sonicated for 10 minutes to give a clear colorless solution.Nitrogen gas was then allowed to bubble through the solution. 1.000 NHydrochloric acid (624 μl, 1.5 equivalent) was added to the solution togive pH 1.88. The solution was allowed to equilibrate at 22° C. for 60minutes.

The solution was then titrated against 1.000 N NaOH at 22° C. to reachpH 11.8. For each addition of base, the solution was allowed toequilibrate until a constant pH reading was reached. The volume of thebase added and the pH reading were recorded for each measurement. 137measurements were taken to finish the experiment.

The data set of pH vs. base volume was analyzed using Hyperquad 2000(Version 2.1, Peter Gans, University of Leeds). Given the model: L⁻+H⁺?LH (pKa₁) and LH+H⁺? LH₂ ⁺ (pKa₂), the pKa values of Apo6619 wereoptimized as pKa₁=8.6 and pKa₂=2.5.

EXAMPLE 11 pKa Determination for Apo6617 by Spectrophotometric Titration

The pKa values of ligands can be determined by spectrophotometrictitration when both the conjugated acid and base absorb in theUV-Visible region. In a typical experiment, the sample solution wasprepared by the following method: Apo6617 (0.792 mg) was weighed into an80-ml beaker, followed by the addition of 0.1 M NaCl (50 ml). Themixture was sonicated for 5 minutes to give a clear colorless solution.Nitrogen gas was allowed to bubble through the solution. 1.000 N NaOH(50 μl) was added to give pH 10.9. The solution was allowed toequilibrate at 22° C. for 1 hour. A sipper system was used for thecirculation of the sample solution between the beaker and the flow cell.

The sample solution was titrated against standard hydrochloric acidsolutions at 22° C. to reach pH 1.40. After each addition of acid thesolution was allowed to equilibrate until a constant pH reading wasreached. The pH and the UV-Vis spectrum were recorded for eachmeasurement. The peak wavelengths of the deprotonated species (L⁻), theneutral species (LH), and the protonated species (LH₂ ⁺) were 314 nm,281 nm, and 249 nm, respectively. In the region of pH>6, after eachaddition of acid there was a slight decrease in the absorbance at 314 nmand a slight increase at 281 nm in each spectrum, whereas in the regionof pH<5, after each addition of acid there was a slight decrease in theabsorbance at 281 nm and a slight increase at 249 nm in each spectrum.The solution was titrated until there was no obvious change in thespectra after several subsequent additions of acid. 116 measurementswere taken to finish the experiment.

The resulting data set was then analyzed using pHAB (Peter Gans,University of Leeds). The pKa values of Apo6617 were optimized aspKa₁=8.6 and pKa₂=2.5.

EXAMPLE 12 Stoichiometry of Fe-Apo6622 Complexes by Job's Method

In a typical experiment, Fe-Apo6622 complex solutions were prepared bymixing a stock solution of Fe³⁺ (atomic absorption standard, 1005 μg/mlin 1 wt. % HCl, Aldrich) and a stock solution of Apo6622 (6.98×10⁻³ M in0.1 M MOPS pH 7.4). 12 sample solutions were prepared. While the sum ofthe total iron concentration (C_(total) ^(iron)) and the total ligandconcentration (C_(total) ^(L)) in each of the 12 sample solutions waskept constant (8.00×10⁻⁴ M), the molar fraction of the ligand, a(a=C_(total) ^(L)/(C_(total) ^(L)+C_(total) ^(iron)), for the 12 samplesolutions were different and were prepared as 0, 0.1, 0.2, 0.3, 0.4,0.5, 0.6, 0.7, 0.75, 0.8, 0.9, and 1.0, respectively. The total volumefor each of the 12 sample solutions was 5 ml, using MOPS (0.1 M, pH 7.4)as the solvent. The pH of the 12 solutions was adjusted by adding NaOHto pH 7.4. The sample solutions were vortexed at room temperature for 3hours, and then placed in a Dubnoff Metabolic Shaking Incubator at 25°C. and at 90 RPM overnight. The sample solutions were centrifuged at4000 rpm for 15 minutes, and then placed back in the incubator at 25° C.without shaking. The UV-Vis spectrum was recorded at 25° C. for each ofthe 12 solutions.

A Job's plot was created with the absorbance at 450 nm as the y-axis anda as the x-axis. A maximum absorbance was found at a=0.75, whichcorresponds to an iron: ligand ratio of 1:3 in the complexes. The Job'splot result is shown in FIG. (1).

Proceeding in a similar manner, the Job's plots of Fe-Apo6617 andFe-Apo6619 were created. They are shown in FIGS. (2) and (3).

EXAMPLE 13 Distribution Coefficient Determination

MOPS buffer (50 mM, pH=7.4) and 1-octanol were used as the aqueous phaseand the organic phase, respectively, for distribution coefficientdeterminations. The MOPS buffer and 1-octanol were pre-saturated witheach other before use. In a typical experiment, an organic stocksolution of Apo6618(1-cyclopropyl-3-hydroxy-6-methyl-4-oxo-1,4-dihydro-pyridine-2-carboxylicacid cyclopropylamide) was prepared by weighing out 0.50 mg of thecompound into a 10-mL volumetric flask and bringing to volume with1-octanol. The solution was then sonicated for 60 minutes so that thesample could dissolve completely. The concentration of the stocksolution was calculated as C⁰ _(org)=2.0×10⁻⁴M. The organic standardsolution of

Apo6618 with 2.0×10⁻⁵ M was prepared in a 10-mL volumetric flask by 10times dilution of the stock solution with 1-octanol. The sample solutionwas prepared in a 10-mL volumetric flask The stock sample solution (3ml) was pipetted into the flask followed by the addition of MOPS buffer(3 ml). The standard and sample solutions were then vortexed for 2hours. After vortexing, the solutions were transferred to test tubes andcentrifuged at 4000 rpm for 15 minutes. UV-Vis spectra were recorded forthe standard solution and the organic (top) phase of the sample solutionat 22° C. The distribution coefficient, D_(7.4), was calculated usingthe following equation:D _(7.4)=[A_(org)/(C⁰ _(org) e _(org)−A_(org))]×(V_(aqu)/V_(org))

Where e_(org)=the molar extinction coefficient of the peak wavelength(?_(max)) obtained from the UV-Vis spectrum of the organic standardsolution; A_(org)=absorbance of the organic phase in the sample solutionat the same ?_(max); C⁰ _(org)=the concentration of the stock solution;V_(aqu)=the volume of MOPS buffer in the sample solution; V_(org)=thevolume of the stock solution in the sample solution.

EXAMPLE 14 Determination of Metal Complexation Constants

A. Instrumental and Chemicals:

A pH meter (Accumet Research AR15, 13-636-AR15, Fisher) and acombination electrode (Accumet Standard-size Glass CombinationElectrode, 13-620-285, Fisher) were used for pH measurements. Beforeusing, the electrode was calibrated with three standard buffer solutions(pH 4.00, pH 7.00, and pH 10.00, Fisher). The titrant was added manuallyby using digital pipettes (Eppendorf). An UV-visible spectrophotometer(Agilent 8453) was used for UV-Vis absorbance measurements.

A sipper system (89068D Agilent) was used whenever pH-dependentabsorbencies were measured. A vortexer (VX-2500 Multi-tube Vortexer, VWRScientific Products) was used for the preparation of sample solutions inboth distribution coefficient and Job's plot experiments.

The metal stock solutions were purchased from Aldrich: Iron atomicabsorption standard solution (1000 μg/ml of Fe in 1 wt. % HCl); Aluminumatonic absorption standard solution (1000 μg/ml of Al in 1 wt % HCl);Calcium atomic absorption standard solution (1000 μg/ml of Ca in 1 wt. %HCl); Copper atomic absorption standard solution (1000 μg/ml of Cu in 1wt. % HNO₃); Magnesium atomic absorption standard solution (1000 μg/mlof Mg in 1 wt. % HNO₃); Manganese atomic absorption standard solution(1000 μg/ml of Mn in 1 wt. % HNO₃); Zinc atomic absorption standardsolution (1000 μg/ml of Zn in 1 wt. % HCl). The standard SodiumHydroxide and Hydrochloric acid solutions were purchased from VWRScientific Products. MOPS (3-[N-Morpholino]propanesulfonic acid) waspurchased from Sigma-Aldrich.

B. Determination of stepwise formation constants for Fe-Apo6619 systemby spectrophotometric titration. Apo6619 is1-cyclopropyl-3hydroxy-6-methyl-4-oxo-1,4-dihydro-pyridine-2-carboxylicacid methylamide.

Stepwise formation constants for M^(n+)-ligand systems were determinedby spectrophotometric titration when metal complexes have a strongabsorbance in the visible region due to ligand to metal charge transfer.In a typical experiment, the sample solution was prepared according tothe following method: Apo6619 (10.7 mg) was weighed into an 80-mlbeaker, followed by the addition of 0.1 M NaCl (50 ml). The mixture wassonicated for 10 minutes to give a clear colorless solution. Iron stocksolution (atomic absorption standard, Aldrich, 496 μl, 8.93E-06 moles)was pipetted into the solution followed by the addition of 1.000 N NaOH(137 μl). The molar ratio between the total iron and the total Apo6619was 1:5.4. The mixture was allowed to equilibrate at room temperatureovernight. Nitrogen was allowed to bubble through the solution. 1.000 NHydrochloric acid (3 ml) was then added to the solution to give pH 1.5.The solution was allowed to equilibrate at 22° C. for 3 hours.

A sipper system was used for the circulation of the sample solutionbetween the beaker and the flow cell.

The sample solution was titrated against standard NaOH solutions at 22°C. to reach pH 6.89. After each addition of base the solution wasallowed to equilibrate until a constant pH reading was reached. The pHand the UV-Vis spectrum were recorded for each measurement. For eachmeasurement enough base was added so that there was a slight increase inthe absorbance of the spectrum. The solution was titrated until therewas no obvious increase in the spectra after several subsequentadditions of base. Altogether 64 measurements were taken to finish theexperiment.

The resulting data set was then analyzed using pHAB. Given the model:L⁻+H⁺? LH (pKa₁), LH+H⁺? LH₂ ⁺ (pKa₂), Fe³⁺+L⁻? FeL²⁺ (K₁), FeL²⁺+L⁻?FeL₂ ⁺ (K₂), FeL₂ ⁺+L⁻? FeL₃ (K₃), and β₃=K₁K₂K₃, the stepwise formationconstants for Fe-Apo6619 system were optimized as log K₁=12.5(1); logK₂=11.6(1); log K₃=9.5(1); log β₃=33.6(2).

C. Determination of stepwise formation constants for Al-Apo6619 systemby potentiometric titration.

Stepwise formation constants for M^(n+)-ligand system were determined bypotentiometric titration when metal complexes (=0.002 M) do notprecipitate during titration. In a typical experiment, the samplesolution was prepared by the following method: Apo6619 (31.91 mg) wasweighed into a 25-ml beaker followed by the addition of 0.1 M NaCl (18.9ml). The mixture was sonicated for 10 minutes to give a clear colorlesssolution. Aluminum stock solution (atomic absorption standard, Aldrich,971 μl, 3.59×10⁻⁵ mole) was pipetted into the solution followed by theaddition of 1.000 N NaOH (229 μl) to give pH 8.56. The molar ratiobetween the total Aluminum and the total Apo6619 was 1:4. For M²⁺metals, a molar ratio of 1:3 was used. Nitrogen was allowed to bubblethrough the solution. The mixture was allowed to equilibrate at 22° C.for 2 hours. 1.000 N Hydrochloric acid (264 μl) was then added to thesolution to give pH 2.20. The solution was allowed to equilibrate at 22°C. for 1 hour.

The solution was titrated against 1.000 N NaOH at 22° C. to reach pH11.0. For each addition of base, the solution was allowed to equilibrateuntil a constant pH reading was reached. The volume of the base addedand the pH reading were then recorded for each measurement. 93measurements were used in the experiment.

The data set of pH vs. base volume was analyzed using Hyperquad 2000.Given the model: L⁻+H⁺? LH (pKa₁), LH+H⁺? LH₂ ⁺ (pKa₂), Al³⁺+L⁻? AlL²⁺(K₁), AlL²⁺+L⁻? AIL₂ ⁺ (K₂), AIL₂ ⁺+L⁻? AlL₃ (K₃), and β₃=K₁K₂K₃, thestepwise formation constants for Al-Apo6619 system were optimized as logK₁=12.6(2); log K₂=9.2(1); log K₃=8.4(1); log β₃=30.2(2).

Calculation of pM^(n+)

pM^(n+) is defined as −log[M(H₂O)_(m)]^(n+) at physiological conditions,i.e.: pH 7.4, a ligand concentration of 10 μM, and a metal concentrationof 1 μM. To calculate pM^(n+) for a ML_(n) system, β_(n) and pKa valuesare needed (β_(n) are the formation constants for M^(n+)+n L⁻? ML_(n);pKa are the equilibrium constants for L⁻+n H⁺? LH_(n) ^((n-1)+)). ThepM^(n+) can be calculated by using Hyss software (Hyperquad Stimulationand Speciation software: HYSS2© 2000 Protonic Sofware).

The data obtained from the above determinations for compounds of formulaI can be found in Table 1 and 2.

EXAMPLE 15 Evaluation of Compounds of Formula I in Iron Overloaded Rats

Effectiveness of Apo6619 and Apo6617 in Promoting Urinary and Fecal IronExcretion in the Iron Overloaded Rat.

The purpose of this study was to determine the effectiveness of Apo6619and Apo6617 in promoting iron excretion in the iron overloaded ratmodel. Iron overloading was achieved by administration of iron dextran.Iron overloading using iron dextran has previously been used to assesschelator efficacy in mice (Kontoghiorghes G. J., Mol Pharmacol. 1986,30(6), 670-3; Bartfay et al., Cardiovasc Res. 1999, 43(4), 892-900),gerbils (Hershko et al., J. Lab Clin Med 2002, 139, 50-58), rats (RakbaN. Biochem Pharmacol. 1998, 55(11):1796-1806) and primates (Bergeron et.al., Blood, 1992, 79(7), 1882-1890). The iron loading regime used inthis study results in a 20-fold increase in liver iron and a 3.8-foldincrease in cardiac iron levels in male rats. Previous studies in thismodel have demonstrated that this model is not associated withsignificant abnormalities in animal weight gain, food consumption,clinical chemistor hematology parameters.

Experimental Protocol:

Six male Sprague-Dawley rats (weighing between 200-250 gms) werereceived from Charles River Laboratories, Montreal, Quebec, Canada. Ratswere iron loaded by administration of iron dextran intraperitoneally ata dose of 100 mg/kg, twice weekly for a period of 4 weeks for a total of8 injections (iron dextran, Sigma). The total volume of iron dextraninjected was 1 mL/kg. Following an eight week period, rats weretransferred to metabolic cages (one rat/cage). Once the animals were inthe metabolic cages, excreta (both urine and feces) were collected dailyfor at least 3 day prior to and 4 days following the administration ofeach chelator. Each of the two chelators (Apo6619 and Apo6617) wasadministered consecutively. Chelators were administered as a single doseof 450 μmoles/kg by oral gavage at a dose volume of 2-4 mL/kg. Theanimals were weighed prior to dosing to enable exact dosageadministration. Animals were checked daily (eyes, skin and movement)after chelator administration to determine if there were any obvioussigns of ill health. Urine and feces were stored at −20° C. untilanalysis for total iron concentrations.

Animal Diet, Water and Housing:

Rats were housed in a climate and light controlled environment(temperature: 19-25° C., relative humidity 40%, 12 hrs light/dark cycle)throughout the study. During the acclimatization, iron loading andequilibration phases, rats were placed in standard cages (2 rats/cage),fed standard rodent chow and given regular tap water ad libitum. Ratswere transferred to metabolic cages (one rat/cage, Nalgene, RochesterN.Y.) after iron loading and equilibration. Three days before placementof the rats in the metabolic cages, rats were fed a low iron diet (3 ppmiron, Dyets Inc., Bethlehem, Pa.) and given Millipore water ad libitum.Rats were continued on the low iron diet for the duration of the study.The purpose of placing animals on a low iron diet was to reduce thebackground noise produced by dietary iron in the fecal samples.

Preparation of Dosing Solution of Chelators:

A 50 mg/ml dosing solution of the chelator of formula I (570 mg) wasfirst dissolved in a mixture of Millipore water (2 ml) and 6N HCl (0.4ml) and brought up to the final volume (11.4 ml) with Millipore water (9ml). Final pH of the solutions was adjusted to pH 4 with diluted sodiumhydroxide solution. Solutions were protected from light and preparedfreshly prior to each administration.

Iron Determinations:

Urine and feces samples were shipped to the Trace Elements Laboratory atthe London Health Sciences Center, London, Ontario, Canada for analysisof total iron concentration. Briefly, feces samples were prepared byadding water, heating to 98° C., vortexing and subsequently freezedrying. The samples were then mixed, and a representative subsampletaken and digested with boiling HNO₃ and H₂O₂. Feces samples were thendiluted 1:100 with ultrapure water prior to running using a highresolution sector field ICP-MS (Finnigan Element 1). Urine samples weredigested with 0.1% HNO₃ and diluted 1/10 prior to running on the ICP-MS.Appropriate calibration curves using iron spiked samples and NISTtraceable standards were used. Samples lying above or below thequantification range were re-run. Since the total amount of urine andfeces produced over a given period of time was known as well as ratweight, total iron levels in the urine and feces are expressed asμg/day/kg. Statistical comparisons within and between groups was madeusing unpaired t-tests. A p value of <0.05 was accepted as significant.

Results

The rats showed no obvious signs of ill health following administrationof any of the chelators. All animals continued to gain weight normallyafter each of the chelators was administered.

Urinary Excretion:

The effectiveness of Apo6619 and Apo6617 in promoting urinary ironexcretion is presented in FIG. 6, below. Baseline urinary excretion, asmeasured during the 3 days prior to Apo6619 administration was 6±1μg/day/kg.

This increased to 240±131 μg/day/kg one day after Apo6619 administration(p=0.007). Excretion subsequently declined to 16±5 μg/day/kg the secondday after Apo6619 administration, however even these levels were stillsignificantly higher than baseline (p=0.004). By the third day, urinaryiron excretion had returned to baseline levels (5±1 μg/day/kg). Apo6617also resulted in increased iron excretion one and two days afteradministration (164±55 and 17±13 μg/day/kg, respectively). Although theurinary excretion produced by Apo6619 was numerically greater than thatachieved with Apo6617 (240±131 μg/day/kg versus 164±55 μg/day/kg, oneday after chelator administration), this difference did not achievestatistical significance due to the fact that one of the six ratsexhibited higher urinary excretion with Apo6617 than Apo6619.

For comparative purposes, deferiprone was also studied at a dose of 450moles/kg in the above model but in a different set of rats (n=6). Thebaseline urinary iron levels measured 9±3 μg/day/kg. These wereincreased to 80±32 μg/day/kg one day after deferiprone administration(p=0.06) and levels returned to baseline by the second day.

Fecal Excretion:

The effectiveness of Apo6619 and Apo6617 in promoting fecal ironexcretion is presented in Table 3. Both the baseline values as well asthe post-chelator induced values represent the sum of iron excreted inthe three days prior to and following chelator administration,respectively. Both Apo6619 and Apo6617 increased fecal iron excretion at450 μmoles/kg, but this reached statistical significance only in theApo6617 group (Apo6619: 4154±1245 μg/day/kg, p=0.08 versus baseline;Apo6617 4411±790 μg/day/kg versus baseline, p=0.008). In a previousstudy in the same model, deferiprone administered to six rats at a doseof 450 moles/kg resulted in fecal iron excretion values of 2157±169μg/day/kg three days after chelator administration.

Second Rat Study at 113μ moles/kq:

A second study was conducted to confirm the efficacy results of Apo6619and Apo6617 observed in the above study and to further characterize theefficacy of these compounds at doses lower than 450 μmol/kg. The studywas conducted in two separate groups of iron overloaded rats. The methodof iron overloading, preparation of dosing solutions and assessment ofefficacy in these rats was similar to that described in the above study.

The first group of rats (n=6) were treated with Apo6619 consecutively atdoses of 28, 113 and 450 μmoles/kg. Similarly, the second group of rats(also n=6) was treated with Apo6617 at these same three doses. A summaryof the excretion data is shown in Table 4. Similar to the previousstudy, both Apo6619 and Apo6617 produced an increase in urinary ironexcretion at the 450 μmoles/kg dose (Apo6619: 11±3 at baseline to 335±761-day post-Apo6619, p=0.0001; Apo6617: 14±4 at baseline to 183±20 1-daypost-Apo6617, p=0.0003). In contrast to the previous study where nosignificant difference between the urinary efficacy of Apo6619 andApo6617 was observed at 450 μmoles/kg, in this study it was clear thatApo6619 was more effective than Apo6617 (p=0.004) at this dose.Similarly, at 113 μmol/kg (25 mg/kg), both Apo6619 and Apo6617 increasedurinary excretion (p<0.005), but Apo6819 was more effective than Apo6617(p=0.01) At 28 μmoles/kg, only Apo6617 produced an increase in ironexcretion (p=0.01) above baseline. However, the magnitude-of theincreased excretion was small for both Apo6619 and Apo6617.

Fecal excretion was increased by Apo6619 at 450 μmoles/kg (p=0.03) andthere was a trend towards increased excretion with Apo6617 as well(p=0.08). No significant increases in fecal excretion were detectablewith either chelator at doses lower than 450 μmoles/kg.

Collectively, both studies show that Apo6619 and Apo6617 result inincreased urinary and fecal iron excretion. This excretion is superiorto that observed in historical studies with deferiprone in the samemodel. While Apo6619 produces significantly greater urinary ironexcretion as compared to Apo6617 at high doses, the superiority ofApo6619 over Apo6617 in producing increased fecal iron excretion was notevident from these studies. In large part, this is due to both the high,and highly variable “background” levels of iron in the feces (i.e. lowsignal to noise ratio) making chelator induced increases in ironexcretion difficult to detect.

EXAMPLE 16 A. Preparation of Fe(Apo6617)₃ Chelate

A pH 9.7 carbonate buffer was prepared by dissolving 0.84 g of sodiumbicarbonate, 1.06 g of sodium carbonate in de-ionized water and dilutingthe solution to 50 ml. Apo6617 (1.028 g, 4.62 mmol) was added to thecarbonate buffer (25 ml). The heterogeneous mixture was stirred for 15minutes at room temperature to give a clear solution. Anhydrous ferricchloride (0.2417 g, 1.49 mmol) was added in small portions over 5 min.at room temperature to give a dark red solution. The flask was thensealed with a septum cap and stirred at room temperature for 42 h.Acetonitrile (30 ml) was added and the solvent was evaporated underreduced pressure to give a dry red mass. The solid was dissolved indichloromethane (90 ml), dried over anhydrous Na₂SO₄, filtered andconcentrated. The crude product was subjected to purification by flashchromatography using elution gradient (dichloromethane/methanol mixture:95/5, 90/10, 85/15 and 80/20). A red solid (900 mg) was obtained. Thesolid was mixed with the mixture of ethyl acetate/methanol (90/10, 60ml) and stirred at RT for 1 h. The insoluble particulate was filteredand the filtrate evaporate under reduce pressure to give the product(800.5 mg). MS (m/z): 742.6 (M⁺+Na), 720.4 (M⁺+1), 634.6, 499.0,469.6,360.4, 334.3

A saturated solution of the Fe(Apo6617)₃ was prepared by dissolving 0.2gm of the material in dichloromethane. The insoluble particulate wasfiltered. 0.3 ml of ethyl acetate was added to 1 ml of the saturateddichloromethane solution in a vial. The vial was capped at roomtemperature. The dark brown crystals were removed for crystallographydetermination. The 3-dimension single crystal structure is shown in FIG.7. The crystallographic data is shown in Table 5 to 6.

B. Preparation of Fe(Apo6619)₃

Apo6619 (4.4488 g, 20.0 mmole) was weighed out into a 100-ml 1-neckround bottom flask equipped with a magnetic stir bar. Deionized water(30 ml) was added to give a suspension. To the mixture was added NaOHsolution (3.336 ml of 6.000 N solution, 20.0 mmole) at room temperatureto give a clear orange-red solution. FeCl₃.6H₂O (1.7735 g, BDH, 97-102%,6.56 mmole) was weighed out into a 30-ml test tube. Deionized water (4ml) was added into the test tube. The mixture was vortexed to give aclear yellow solution. The FeCl₃ solution was added to the above Apo6619solution dropwise. The mixture was stirred at room temperature for 6days. Solid was formed at this time. The solid was collected by suctionfiltration. The solid was transferred back to the round bottom flask. 50ml of acetone and 3 ml of deionized water was added. The mixture wasstirred for a few hours. The solid was then collected by suctionfiltration. The solid was air dried to give 3.1 g (yield=66%). MS: 720.6(M+1). Single crystals of Fe(Apo6619)₃ were grown from diffusion oftoluene into wet DMF. The X-ray crystal structure of Fe(Apo6619)₃ isshown in FIG. 8.

EXAMPLE 17 Determination of E₁₁₂ of Fe-Apo6619 System

A. Materials & Instruments

Potassium ferricyanide (III) was purchased from Aldrich. Deferoxaminemesylate (DFO) was purchased from Sigma. Iron atomic absorption standardsolution (contains 1005 μg/mL of Fe in 1 wt. % HCl) was purchased fromAldrich. Electrochemical measurements were performed with a cyclicvoltammetric analyzer (BAS, CV-50W Potentiostat). Software BAS CV-50WVersion 2.31 was used. The following electrodes were used fordetermining redox potentials of the iron complexes: Ag/AgCl referenceelectrode (BAS, MF-2052); platinum auxiliary electrode (BAS, MW-1032);and glassy carbon working electrode (BAS, MF-2012). A pH meter (AccumetResearch AR15, 13-636-AR15, Fisher Scientific) and pH electrode(AccupHast combination electrode, 13-620-297, Fisher Scientific) wereused for pH adjustment of the sample solutions.

B. Preparation of Sample Solutions

2.0 mM solution of Fe(DFO) in 0.1 M NaCl (pH 7.4)

148.1 mg of Deferoxamine mesylate (purity=95%) was accurately weighedout into a 100-mL volumetric flask. The solid was dissolved in about 30mL of 0.1 M NaCl to give a clear colorless solution. To the solution wasadded 11.114 mL of the standard iron solution (contains 1005 μg/mL of Fein 1 wt. % HCl). The solution was diluted with 0.1 M NaCl to the 100 mlmark in the volumetric flask. The resulting solution was vortexed toensure complete mixing. The solution was transferred to a 200-mL beaker.The pH of the solution was then adjusted to about 7.1 by adding standardsolutions of sodium hydroxide. The beaker was then covered with parafilmand the solution was left stirring overnight. The pH of the solution wasadjusted to 7.40 in the following test day. The calculated molar ratiobetween irontotal and DFO_(total) is 1:1.07.

2.0 mM solution of Fe(Apo6619)₃ in 0.1 M NaCl (pH 7.4)

70.0 mg of Apo6619 was accurately weighed out into a 50-mL volumetricflask. The solid was dissolved in about 15 mL of 0.1 M NaCl to give aclear colorless solution. To the solution was added 5557 μL of thestandard iron solution (contains 1005 μg/mL of Fe in 1 wt. % HCl). 0.1 MNaCl was then added to diluted the total volume to 50 ml. The resultingsolution was vortexed to ensure complete mixing. The solution wastransferred to an 80-mL beaker. The pH of the solution was then adjustedto about 7.1 by adding standard solutions of sodium hydroxide. Thebeaker was then covered with paraflilm and the solution was leftstirring overnight. The pH of the solution was adjusted to 7.40 in thefollowing test day. The calculated molar ratio between iron_(total) andApo6619_(total) is 1:3.15. In a similar manner, a solution of 2.0 mM ofFe(deferiprone)₃ in 0.1 M NaCl (pH 7.4) was prepared.

C. Determination of Redox Potentials of Iron Complexes

All potentials in the text are given versus the Ag/AgCl referenceelectrode. The redox potentials of 2.0 mM of K₃Fe(CN)₆ in 1.0 Mpotassium nitrate were measured at the beginning of each working day toverify the proper functioning of the cyclic voltammeter. The redox peakpotentials of 2.0 mM solutions of iron complexes at pH 7.4, that is,Fe(DFO), Fe(L1)₃, and Fe(Apo6619)₃, were determined. The samplesolutions of iron complexes were purged with argon for 15 minutes beforeCV scans, and the solution was under argon during measurements. Theglassy carbon working electrode was polished on alumina after each scan.The scan rate used was 300 mV/sec for Potassium ferricyanide (III)solution, and was 450 mV/sec for the solutions of Fe(DFO), Fe(L1)₃, andFe(Apo6619)₃. FIG. 9 shows the cyclic voltammograms of iron(III)L_(n)complexes at pH 7.4: a) K₃Fe(CN)₆; b) Fe(DFO); c) Fe(L1)₃;{L1=deferiprone} and d) Fe(Apo6619)₃. The reduction peak potential(E_(p) ^(red)), the oxidation peak potential (E_(p) ^(ox)), the absolutedifference between E_(p) ^(red) and E_(p) ^(ox) (ΔE_(p)), and redoxpotential (E_(1/2)) of the four iron complexes measured. E_(1/2) valueis calculated as (E_(p) ^(red)+E_(p) ^(ox))/2 is reported in the tablewithin FIG. 9.

The redox potentials of 2.0 mM of K₃Fe(CN)₆ in 1.0 M potassium nitratewere measured at the beginning of each working day to verify the properfunctioning of the cyclic voltammeter. In a typical measurement, theE_(p) ^(red), E_(p) ^(ox), ΔE_(p), and E_(1/2) values of K₃Fe(CN)₆determined in this lab using glassy carbon working electrode are 197 mV,282 mV, 85 mV, and 240 mV, respectively. The values from BioanalyticalSystems Inc. (BAS) using platinum working electrode are 237 mV, 306 mV,69 mV, and 272 mV, respectively. From a theoretical perspective, ΔE_(p)should be about 60 mV for a single electron transfer process. Theexperimental values are considered in good agreement with those fromBAS.

Unlike K₃Fe(CN)₆, the redox properties of Fe(DFO), Fe(L1)₃, andFe(Apo6619)₃ are extremely sensitive to the status of working electrodesurface. The redox potentials were reproducible only after carefulpolishing of the glassy carbon working electrode on alumina after eachscan.

The ΔE_(p) values of Fe(DFO), Fe(L1)₃, and Fe(Apo6619)₃ are 112 mV, 107mV, and 85 mV, respectively. It can be seen clearly (FIG. 9) that thecyclic voltammograms of Fe(DFO), Fe(L1)₃, and Fe(Apo6619)₃ are basicallyreversible. Based on these two observations, it is reasonable to assumethat the cyclic voltammograms of Fe(DFO), Fe(L1)₃, and Fe(Apo6619)₃represent a reversible single electron transfer process for eachcomplex: Fe(III)L_(n) ∴ Fe(II)L_(n). The E_(1/2) value of Fe(DFO)determined in this lab is −698 mV versus the Ag/AgCl referenceelectrode, which is in excellent agreement to literature value (−688 mV)(A. L. Crumbliss et al, Inorganic Chemistry, 2003, 42, 42-50)The E_(1/2)value of Fe(Apo6619)₃ is −691 mV, which is similar to that of Fe(DFO).

The above examples are provided by way of illustration only and are inno way intended to limit the scope of the invention. One of skill in theart will understand that the invention may be modified in various wayswithout departing from the spirit or principle of the invention. Weclaim all such modifications.

The electrochemical properties of iron(III)L_(n) complexes at pH 7.4 arelisted below:

System E_(p) ^(red) (mV) E_(p) ^(ox) (mV) ΔE_(p) (mV) E_(1/2) (mV)K₃Fe(CN)₆ 197 282 85 240 Fe(DFO) −754 −642 112 −698 Fe(deferiprone)₃−887 −780 107 −834 Fe(Apo6619)₃ −733 −648 85 −691

TABLE 1 Chemical Properties of compounds of formula I. QMPR Plus ™software Cal. Human Jejunal Effective Permeability Structure Compound #D_(7.4) pKas Log β₃ pFe³⁺ [cm/s × 10⁻⁴]

CP502 0.04  2.7, 8.5 33.6 20.9 0.81

Apo6617 0.099 2.4, 8.5 33.6 20.8 1.11

Apo6618 0.331 2.5, 8.6 33.6 20.5 1.49

Apo6619 0.109 2.5, 8.6 33.4 20.7 1.11

Apo6620 0.78  2.7, 8.7 33.8 20.3 1.46

Apo6621 2.2  2.5, 8.7 34.3 20.9 1.65

Apo6622 0.357 2.8, 8.6 33.9 20.8 1.28

TABLE 2 Metal ion binding selectivity of Apo6619 (pKa₁ = 2.5, pKa₂ =8.6) log scale Fe(III) Al(III) Cu(II) Zn(II) Mn(II) Mg(II) Ca(II) K₁12.5 9.3 8.9 6.3 5.1 4.1 3 K₂ 11.6 9.5 7.7 5.8 4.3 3.2 2.1 K₃ 9.5 8.2 —— — — — β₂ — — 16.6 12.1 9.4 7.3 5.1 β₃ 33.6 27 — — — — — PM 20.5 13.910 6.4 6.0 6.0 6.0

TABLE 3 Effectiveness of Apo6619 and Apo6617 administered at a dose of450 μmoles/kg in Promoting Fecal Iron Excretion in the Iron OverloadedRats (n = 6). Values are expressed as μg/day/kg. Fecal excretion valuesthree days after putative chelator administration are given. Values areexpressed as mean ± 1 SD. Fecal excretion Test Article (μg/daykg)Baseline 3057 ± 184 Apo6619 4154 ± 1245 Apo6617 4411 ± 790

TABLE 4 Effectiveness of Apo6619 and Apo6617 in Promoting Urinary andFecal Iron Excretion in the Iron Overloaded Rats (n = 6/group). Valuesare expressed as μg/day/kg. Fecal excretion values 3 days after chelatoradministration are given and compared to the baseline values determined3 days prior to chelator administration. Values are expressed as mean ±1 SD. Iron excretion data expressed in μg/day/kg (±SD) Urine (1 daypost-chelator) Feces (3 days)¹ Compound Apo6617 Apo6619 Apo6617 Apo6619Dose Level 0 (Baseline)  14 ± 4  11 ± 3 2300 ± 1003 2575 ± 871  28μmol/kg  24 ± 6*  14 ± 4 Not Not Measured Measured 113 μmol/kg  28 ± 8* 51 ± 15*τ 2411 ± 335 3033 ± 1076 450 μmol/kg 183 ± 20* 335 ± 76*τ 3228± 437 3831 ± 790* ¹Assessment of 3-day fecal excretion was required toallow for transit of the iron through the gastrointestinal tract. *p <0.05 versus baseline value in the same group τp < 0.05 versus Apo6617 atthe same dose

TABLE 5 Crystal data and structure refinement for Fe(Apo6617)₃Identification code Fe(Apo6617)₃ Empirical formula C₃₃H₄₂FeN₆O_(10.50)Formula weight 746.58 Temperature 150(1) K Wavelength 0.71073 Å Crystalsystem Triclinic Space group P-1 Unit cell dimensions a = 10.9760(4) Å α= 94.283(2)°. b = 11.3790(4) Å β = 90.351(2)°. c = 13.9952(5) Å γ =91.731(2)°. Volume 1742.18(11) Å³ Z 2 Density (calculated) 1.423 Mg/m³Absorption coefficient 0.500 mm⁻¹ F(000) 784 Crystal size 0.30 × 0.14 ×0.04 mm³ Theta range for data collection 2.62 to 25.00°. Index ranges−13 <= h <= 13, −13 <= k <= 13, −16 <= l <= 16 Reflections collected16641 Independent reflections 6114 [R(int) = 0.0753] Completeness totheta = 25.00° 99.8% Absorption correction Semi-empirical fromequivalents Max. and min. transmission 0.966 and 0.892 Refinement methodFull-matrix least-squares on F² Data/restraints/parameters 6114/2/462Goodness-of-fit on F² 1.034 Final R indices [I > 2 sigma(I)] R1 =0.0566, wR2 = 0.1410 R indices (all data) R1 = 0.0830, wR2 = 0.1594Extinction coefficient none Largest diff. peak and hole 0.609 and −0.539e · Å⁻³

TABLE 6 Bond lengths [Å] and angles [°] for Fe(Apo6617)₃ Fe(1)—O(5)1.985(2) Fe(1)—O(8) 2.010(2) Fe(1)—O(2) 2.016(2) Fe(1)—O(7) 2.020(3)Fe(1)—O(4) 2.025(2) Fe(1)—O(1) 2.048(2) O(1)—C(1) 1.294(4) O(2)—C(5)1.327(4) O(4)—C(12) 1.299(4) O(5)—C(17) 1.321(4) O(6)—C(20) 1.233(4)O(7)—C(24) 1.297(4) O(8)—C(28) 1.329(4) O(9)—C(31) 1.218(4) N(1)—C(3)1.351(5) N(1)—C(4) 1.397(5) N(1)—C(7) 1.487(5) N(3)—C(14) 1.355(5)N(3)—C(16) 1.390(5) N(3)—C(19) 1.483(5) N(5)—C(26) 1.361(5) N(5)—C(27)1.387(4) N(5)—C(30) 1.474(5) C(1)—C(2) 1.393(5) C(1)—C(5) 1.434(5)C(2)—C(3) 1.383(6) C(3)—C(6) 1.495(5) C(4)—C(5) 1.384(5) C(4)—C(8)1.493(6) C(8)—O(3*) 1.256(7) C(8)—O(3) 1.281(8) C(8)—N(2*) 1.325(8)C(8)—N(2) 1.378(8) N(2)—C(9) 1.608(12) C(9)—C(10) 1.447(16) C(9)—C(11)1.505(13) C(10)—C(11) 1.581(16) N(2*)—C(9*) 1.556(13) C(9*)—C(11*)1.440(12) C(9*)—C(10*) 1.454(15) C(10*)—C(11*) 1.474(14) C(12)—C(13)1.384(5) C(12)—C(17) 1.441(5) C(13)—C(14) 1.393(5) C(14)—C(18) 1.498(5)C(16)—C(17) 1.383(5) C(16)—C(20) 1.508(5) C(20)—N(4) 1.331(5) N(4)—C(21)1.474(10) C(21)—C(23) 1.434(15) C(21)—C(22) 1.492(13) C(22)—C(23)1.392(15) C(21*)—C(22*) 1.461(19) C(21*)—C(23*) 1.535(16) C(22*)—C(23*)1.582(18) C(24)—C(25) 1.395(5) C(24)—C(28) 1.425(5) C(25)—C(26) 1.363(5)C(26)—C(29) 1.504(5) C(27)—C(28) 1.376(5) C(27)—C(31) 1.501(5)C(31)—N(6) 1.310(5) N(6)—C(32) 1.514(12) C(32)—C(34) 1.417(15)C(32)—C(33) 1.485(16) C(33)—C(34) 1.459(15) C(32*)—C(33*) 1.433(16)C(32*)—C(34*) 1.53(2) C(33*)—C(34*) 1.45(2) O(11)—O(11)#1 1.550(16)O(5)—Fe(1)—O(8) 88.36(9) O(5)—Fe(1)—O(2) 88.97(10) O(8)—Fe(1)—O(2)95.35(10) O(5)—Fe(1)—O(7) 166.27(10) O(8)—Fe(1)—O(7) 80.41(10)O(2)—Fe(1)—O(7) 99.86(11) O(5)—Fe(1)—O(4) 81.11(10) O(8)—Fe(1)—O(4)101.01(10) O(2)—Fe(1)—O(4) 160.54(10) O(7)—Fe(1)—O(4) 93.23(10)O(5)—Fe(1)—O(1) 103.01(10) O(8)—Fe(1)—O(1) 167.38(10) O(2)—Fe(1)—O(1)79.61(10) O(7)—Fe(1)—O(1) 89.01(10) O(4)—Fe(1)—O(1) 86.34(10)C(1)—O(1)—Fe(1) 113.1(2) C(5)—O(2)—Fe(1) 114.4(2) C(12)—O(4)—Fe(1)112.7(2) C(17)—O(5)—Fe(1) 113.2(2) C(24)—O(7)—Fe(1) 113.6(2)C(28)—O(8)—Fe(1) 112.7(2) C(3)—N(1)—C(4) 121.3(3) C(3)—N(1)—C(7)117.7(3) C(4)—N(1)—C(7) 120.8(3) C(14)—N(3)—C(16) 121.3(3)C(14)—N(3)—C(19) 118.6(3) C(16)—N(3)—C(19) 120.1(3) C(26)—N(5)—C(27)120.5(3) C(26)—N(5)—C(30) 120.7(3) C(27)—N(5)—C(30) 118.7(3)O(1)—C(1)—C(2) 124.9(4) O(1)—C(1)—C(5) 117.8(3) C(2)—C(1)—C(5) 117.3(3)C(3)—C(2)—C(1) 121.3(4) N(1)—C(3)—C(2) 120.4(3) N(1)—C(3)—C(6) 119.4(4)C(2)—C(3)—C(6) 120.2(4) C(5)—C(4)—N(1) 119.0(3) C(5)—C(4)—C(8) 121.6(3)N(1)—C(4)—C(8) 119.3(3) O(2)—C(5)—C(4) 124.3(3) O(2)—C(5)—C(1) 115.1(3)C(4)—C(5)—C(1) 120.6(3) O(3*)—C(8)—O(3) 26.2(4) O(3*)—C(8)—N(2*)118.8(5) O(3)—C(8)—N(2*) 117.9(6) O(3*)—C(8)—N(2) 117.3(6)O(3)—C(8)—N(2) 127.8(6) N(2*)—C(8)—N(2) 22.3(4) O(3*)—C(8)—C(4) 125.0(4)O(3)—C(8)—C(4) 119.1(5) N(2*)—C(8)—C(4) 116.2(4) N(2)—C(8)—C(4) 112.9(4)C(8)—N(2)—C(9) 106.3(6) C(10)—C(9)—C(11) 64.7(7) C(10)—C(9)—N(2)103.8(9) C(11)—C(9)—N(2) 113.2(8) C(9)—C(10)—C(11) 59.4(7)C(9)—C(11)—C(10) 55.8(7) C(8)—N(2*)—C(9*) 119.2(7) C(11*)—C(9*)—C(10*)61.2(7) C(11*)—C(9*)—N(2*) 114.6(8) C(10*)—C(9*)—N(2*) 100.4(9)C(9*)—C(10*)—C(11*) 58.9(7) C(9*)—C(11*)—C(10*) 59.8(7) O(4)—C(12)—C(13)125.6(3) O(4)—C(12)—C(17) 116.3(3) C(13)—C(12)—C(17) 118.0(3)C(12)—C(13)—C(14) 120.9(3) N(3)—C(14)—C(13) 120.2(3) N(3)—C(14)—C(18)119.4(3) C(13)—C(14)—C(18) 120.5(4) C(17)—C(16)—N(3) 119.7(3)C(17)—C(16)—C(20) 120.0(3) N(3)—C(16)—C(20) 120.3(3) O(5)—C(17)—C(16)123.9(3) O(5)—C(17)—C(12) 116.5(3) C(16)—C(17)—C(12) 119.7(3)O(6)—C(20)—N(4) 124.1(4) O(6)—C(20)—C(16) 122.9(3) N(4)—C(20)—C(16)113.0(3) C(20)—N(4)—C(21) 126.2(5) C(23)—C(21)—N(4) 120.5(8)C(23)—C(21)—C(22) 56.8(7) N(4)—C(21)—C(22) 118.0(7) C(23)—C(22)—C(21)59.5(7) C(22)—C(23)—C(21) 63.7(8) C(22*)—C(21*)—C(23*) 63.7(8)C(21*)—C(22*)—C(23*) 60.4(8) C(21*)—C(23*)—C(22*) 55.9(8)O(7)—C(24)—C(25) 125.8(3) O(7)—C(24)—C(28) 116.6(3) C(25)—C(24)—C(28)117.5(3) C(26)—C(25)—C(24) 121.9(4) N(5)—C(26)—C(25) 120.1(3)N(5)—C(26)—C(29) 118.4(3) C(25)—C(26)—C(29) 121.5(4) C(28)—C(27)—N(5)120.4(3) C(28)—C(27)—C(31) 120.9(3) N(5)—C(27)—C(31) 118.6(3)O(8)—C(28)—C(27) 123.8(3) O(8)—C(28)—C(24) 116.6(3) C(27)—C(28)—C(24)119.5(3) O(9)—C(31)—N(6) 123.8(4) O(9)—C(31)—C(27) 121.5(3)N(6)—C(31)—C(27) 114.7(3) C(31)—N(6)—C(32) 119.2(5) C(34)—C(32)—C(33)60.3(8) C(34)—C(32)—N(6) 126.8(9) C(33)—C(32)—N(6) 118.8(8)C(34)—C(33)—C(32) 57.5(7) C(32)—C(34)—C(33) 62.2(7) C(33*)—C(32*)—C(34*)58.7(9) C(32*)—C(33*)—C(34*) 64.0(9) C(33*)—C(34*)—C(32*) 57.3(8)Symmetry transformations used to generate equivalent atoms: #1 −x, −y +1, −z

TABLE 7 Crystal data and structure refinement for Fe(Apo6619)₃Identification code Fe(Apo6619)₃ Empirical formula C37.50 H53.50 FeN7.50 O12.50 Formula weight 865.23 Temperature 150(1) K Wavelength0.71073 Å Crystal system Triclinic Space group P-1 Unit cell dimensionsa = 11.9319(8) Å α = 116.811(3)°. b = 14.3968(9) Å β = 108.353(3)°. c =15.3024(9) Å γ = 95.164(4)°. Volume 2141.6(2) Å³ Z 2 Density(calculated) 1.342 Mg/m³ Absorption coefficient 0.421 mm⁻¹ F(000) 914Crystal size 0.22 × 0.21 × 0.10 mm³ Theta range for data collection 2.62to 27.59°. Index ranges −15 <= h <= 14, −18 <= k <= 18, −16 <= l <= 19Reflections collected 20782 Independent reflections 9756 [R(int) =0.0469] Completeness to theta = 27.59° 98.4% Absorption correctionSemi-empirical from equivalents Max. and min. transmission 0.949 and0.805 Refinement method Full-matrix least-squares on F²Data/restraints/parameters 9756/0/518 Goodness-of-fit on F² 1.053 FinalR indices [I > 2 sigma(I)] R1 = 0.0582, wR2 = 0.1519 R indices (alldata) R1 = 0.0928, wR2 = 0.1663 Extinction coefficient 0.0061(14)Largest diff. peak and hole 0.559 and −0.504 e · Å⁻³

TABLE 8 Bond lengths [Å] and angles [°] for Fe(Apo6619)₃. Fe(1)—O(5)1.9725(19) Fe(1)—O(3) 2.0180(17) Fe(1)—O(4) 2.0185(19) Fe(1)—O(6)2.0300(18) Fe(1)—O(2) 2.0320(18) Fe(1)—O(1) 2.0634(17) O(1)—C(1)1.312(3) O(2)—C(2) 1.292(3) O(3)—C(12) 1.314(3) O(4)—C(13) 1.294(3)O(5)—C(23) 1.323(3) O(6)—C(24) 1.295(3) O(7)—C(10) 1.250(3) O(8)—C(21)1.244(3) O(9)—C(32) 1.249(3) N(1)—C(4) 1.362(4) N(1)—C(5) 1.410(3)N(1)—C(7) 1.466(4) N(2)—C(15) 1.359(4) N(2)—C(16) 1.393(3) N(2)—C(18)1.465(3) N(3)—C(26) 1.373(4) N(3)—C(27) 1.382(3) N(3)—C(29) 1.469(4)N(4)—C(10) 1.329(4) N(4)—C(11) 1.464(4) N(5)—C(21) 1.319(4) N(5)—C(22)1.449(4) N(6)—C(32) 1.317(4) N(6)—C(33) 1.466(4) C(1)—C(5) 1.394(4)C(1)—C(2) 1.442(4) C(2)—C(3) 1.392(4) C(3)—C(4) 1.383(4) C(4)—C(6)1.498(4) C(5)—C(10) 1.485(4) C(7)—C(8) 1.480(5) C(7)—C(9) 1.481(5)C(8)—C(9) 1.500(6) C(12)—C(16) 1.370(4) C(12)—C(13) 1.438(4) C(13)—C(14)1.393(4) C(14)—C(15) 1.387(4) C(15)—C(17) 1.494(4) C(16)—C(21) 1.512(4)C(18)—C(19) 1.486(4) C(18)—C(20) 1.493(4) C(19)—C(20) 1.488(5)C(23)—C(27) 1.377(4) C(23)—C(24) 1.438(4) C(24)—C(25) 1.398(4)C(25)—C(26) 1.372(4) C(26)—C(28) 1.500(4) C(27)—C(32) 1.494(4)C(29)—C(31) 1.480(5) C(29)—C(30) 1.481(4) C(30)—C(31) 1.494(5)N(1S)—C(2S) 1.345(6) N(1S)—C(1S) 1.367(7) N(1S)—C(3S) 1.442(5)O(1S)—C(2S) 1.253(6) O(5)—Fe(1)—O(3) 90.66(7) O(5)—Fe(1)—O(4) 161.25(7)O(3)—Fe(1)—O(4) 80.33(7) O(5)—Fe(1)—O(6) 80.93(7) O(3)—Fe(1)—O(6)106.44(7) O(4)—Fe(1)—O(6) 85.88(7) O(5)—Fe(1)—O(2) 93.91(8)O(3)—Fe(1)—O(2) 85.84(7) O(4)—Fe(1)—O(2) 101.72(8) O(6)—Fe(1)—O(2)166.63(7) O(5)—Fe(1)—O(1) 103.56(7) O(3)—Fe(1)—O(1) 159.41(7)O(4)—Fe(1)—O(1) 89.78(7) O(6)—Fe(1)—O(1) 90.66(7) O(2)—Fe(1)—O(1)78.50(7) C(1)—O(1)—Fe(1) 113.92(16) C(2)—O(2)—Fe(1) 115.03(16)C(12)—O(3)—Fe(1) 112.01(16) C(13)—O(4)—Fe(1) 112.70(16) C(23)—O(5)—Fe(1)113.72(16) C(24)—O(6)—Fe(1) 112.55(16) C(4)—N(1)—C(5) 121.5(2)C(4)—N(1)—C(7) 118.7(2) C(5)—N(1)—C(7) 119.7(2) C(15)—N(2)—C(16)121.5(2) C(15)—N(2)—C(18) 120.4(2) C(16)—N(2)—C(18) 118.1(2)C(26)—N(3)—C(27) 121.0(2) C(26)—N(3)—C(29) 119.8(2) C(27)—N(3)—C(29)119.2(2) C(10)—N(4)—C(11) 121.8(2) C(21)—N(5)—C(22) 121.9(2)C(32)—N(6)—C(33) 120.9(3) O(1)—C(1)—C(5) 124.9(2) O(1)—C(1)—C(2)115.5(2) C(5)—C(1)—C(2) 119.3(2) O(2)—C(2)—C(3) 124.8(3) O(2)—C(2)—C(1)116.8(2) C(3)—C(2)—C(1) 118.4(2) C(4)—C(3)—C(2) 121.1(3) N(1)—C(4)—C(3)120.0(2) N(1)—C(4)—C(6) 119.6(2) C(3)—C(4)—C(6) 120.3(3) C(1)—C(5)—N(1)118.5(2) C(1)—C(5)—C(10) 122.0(2) N(1)—C(5)—C(10) 118.7(2)N(1)—C(7)—C(8) 118.2(3) N(1)—C(7)—C(9) 119.6(3) C(8)—C(7)—C(9) 60.9(3)C(7)—C(8)—C(9) 59.6(3) C(7)—C(9)—C(8) 59.5(2) O(7)—C(10)—N(4) 121.7(3)O(7)—C(10)—C(5) 123.0(3) N(4)—C(10)—C(5) 115.2(2) O(3)—C(12)—C(16)125.1(3) O(3)—C(12)—C(13) 116.0(2) C(16)—C(12)—C(13) 118.8(2)O(4)—C(13)—C(14) 124.3(2) O(4)—C(13)—C(12) 116.9(2) C(14)—C(13)—C(12)118.8(2) C(15)—C(14)—C(13) 120.7(3) N(2)—C(15)—C(14) 119.4(2)N(2)—C(15)—C(17) 120.4(2) C(14)—C(15)—C(17) 120.1(3) C(12)—C(16)—N(2)120.3(2) C(12)—C(16)—C(21) 121.5(2) N(2)—C(16)—C(21) 117.8(2)N(2)—C(18)—C(19) 118.1(2) N(2)—C(18)—C(20) 119.1(2) C(19)—C(18)—C(20)59.9(2) C(18)—C(19)—C(20) 60.3(2) C(19)—C(20)—C(18) 59.8(2)O(8)—C(21)—N(5) 124.0(3) O(8)—C(21)—C(16) 119.9(2) N(5)—C(21)—C(16)116.1(2) O(5)—C(23)—C(27) 124.7(2) O(5)—C(23)—C(24) 115.9(2)C(27)—C(23)—C(24) 119.3(2) O(6)—C(24)—C(25) 125.3(2) O(6)—C(24)—C(23)116.7(2) C(25)—C(24)—C(23) 118.0(2) C(26)—C(25)—C(24) 121.4(3)C(25)—C(26)—N(3) 119.8(2) C(25)—C(26)—C(28) 120.6(3) N(3)—C(26)—C(28)119.6(2) C(23)—C(27)—N(3) 120.5(2) C(23)—C(27)—C(32) 120.8(2)N(3)—C(27)—C(32) 118.3(2) N(3)—C(29)—C(31) 119.3(3) N(3)—C(29)—C(30)118.4(3) C(31)—C(29)—C(30) 60.6(2) C(29)—C(30)—C(31) 59.7(2)C(29)—C(31)—C(30) 59.7(2) O(9)—C(32)—N(6) 123.8(3) O(9)—C(32)—C(27)120.8(2) N(6)—C(32)—C(27) 115.4(3) C(2S)—N(1S)—C(1S) 119.6(5)C(2S)—N(1S)—C(3S) 117.8(4) C(1S)—N(1S)—C(3S) 122.5(5) O(1S)—C(2S)—N(1S)120.9(6) Symmetry transformations used to generate equivalent atoms:

1. A 3-hydroxypyridin-4-one compound of formula I:

wherein: R¹ is X with the proviso that R² is Y; or R¹ is T with theproviso that R² is W; X is C₃-C₆ cycloalkyl; Y is selected from thegroup consisting of C₃-C₆ cycloalkyl, C₁ to C₆ alkyl and C₁ to C₆ alkylmonosubstituted with a C₃-C₆ cycloalkyl; T is C₁ to C₆ alkyl; W is C₃-C₆cycloalkyl; R³ is selected from the group consisting of hydrogen and C₁to C₆ alkyl; R⁴ is selected from the group consisting of hydrogen and C₁to C₆ alkyl; R⁵ is selected from the group consisting of hydrogen and C₁to C₆ alkyl; and/or a pharmaceutically acceptable salt thereof.
 2. Acompound according to claim 1 wherein R¹ is X with the proviso that R²is Y.
 3. A compound of claim 2 wherein X is C₃-C₆ cycloalkyl, Y is C₁ toC₆ alkyl and R⁵ is hydrogen or methyl.
 4. A compound of claim 3 whereinX is cyclopropyl, Y is methyl, R³ is hydrogen, R⁴ is methyl and R⁵ ishydrogen, and wherein said compound is1-cyclopropyl-3-hydroxy-6-methyl-4-oxo-1,4-dihydro-pyridine-2-carboxylicacid methylamide.
 5. A pharmaceutical composition comprising1-cyclopropyl-3-hydroxy-6-methyl-4-oxo-1,4-dihydro-pyridine-2-carboxylicacid methylamide and a pharmaceutically acceptable carrier.
 6. Thepharmaceutical composition of claim 5, which is adopted for oraladministration.
 7. A compound of claim 2 wherein X is C₃-C₆ cycloalkyl,Y is C₃-C₆ cycloalkyl and R⁵ is hydrogen.
 8. A compound of claim 7wherein X is cyclopropyl, Y is cyclopropyl, R³ is hydrogen, R⁴ ismethyl, and wherein said compound isN,1-dicyclopropyl-3-hydroxy-6-methyl-4-oxo-1,4-dihydropyridine-2-carboxamide.9. A compound of claim 3 wherein X is cyclopropyl, Y is methyl, R³ ishydrogen, R⁴ is methyl and R⁵ is methyl, and wherein said compound is1-cyclopropyl-3-hydroxy-N,N,6-trimethyl-4-oxo-1,4-dihydropyridine-2-carboxamide.10. A compound according to claim 1 wherein R¹ is T with the provisothat R² is W.
 11. A compound of claim 10 wherein T is C₁-C₆ alkyl and Wis C₃-C₆ cycloalkyl.
 12. A compound of claim 11 wherein T is methyl, Wis cyclopropyl, R³ is hydrogen, R⁴ is methyl and R⁵ is hydrogen, andwherein said compound is3-hydroxy-1,6-dimethyl-4-oxo-1,4-dihydro-pyridine-2-carboxylic acidcyclopropylamide.
 13. A pharmaceutical composition comprising a compoundaccording to claim 1 and a physiologically acceptable carrier.
 14. Apharmaceutical composition according to claim 13, which is adopted fororal administration.
 15. A method of treating at least one medicalcondition related to a toxic concentration of iron comprisingadministering to an animal suffering from said condition atherapeutically effective amount of the compound of claim 4, whereinsaid at least one medical condition is selected from the groupconsisting of thalassaemia, sickle cell disease and haemochromatosis.